Anti-cd40 antibodies and uses thereof

ABSTRACT

The present invention encompasses antagonist anti-CD40 antibodies and antigen-binding portions thereof. Specifically, the invention relates to humanized anti-CD40 antibodies. In certain embodiments, antibodies of the invention neutralize human CD40 (hCD40) activity. Antibodies, or antibody portions, of the invention are useful for detecting CD40 and for inhibiting CD40 activity, e.g., in a human subject suffering from a disorder in which CD40 activity is detrimental.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/167,598, filed on May 27, 2016, now U.S. Pat. No. 10,174,121,issued Jan. 8, 2019, which claims priority to U.S. ProvisionalApplication No. 62/168,425, filed May 29, 2015, the entire contents ofwhich are hereby incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 21, 2018, isnamed A103017_1040US_D1_SL.txt and is 88,948 bytes in size.

FIELD OF THE INVENTION

The present invention relates to CD40 (CD40) antibodies, andantigen-binding portions thereof, and their use in the prevention and/ortreatment of various diseases.

BACKGROUND OF THE INVENTION

CD40 is a tumor necrosis factor (TNF) receptor family member that playsan important role in B cell development, lymphocyte activation, andantigen presenting cells (APC) function. CD40 expression on epithelium,leukocytes, and vascular endothelium is elevated in organ-specificautoimmune diseases as well as systemic autoimmunity such as systemiclupus erythematosus (SLE). Disruption of the CD40L/CD40 signalingpathway reduces production of proinflammatory cytokines such as IL-23and TNF, reduces T helper cell differentiation and function, andinhibits macrophage activation in patients with chronic inflammatorydiseases such as Crohn's disease. The interaction of CD40 with CD40Linduces both humoral and cell-mediated immune responses. CD40 regulatesthis ligand-receptor pair to activate B cells and otherantigen-presenting cells (APC) including dendritic cells (DCs).

CD40 is a 48 kDa type 1 transmembrane protein (van Kooten, J LeukocBiol. 2000 January; 67(1):2-17) that is expressed on a wide range ofhematopoietic (lymphocytes, monocytes, dendritic cells) andnon-hematopoietic (epithelium, endothelium, fibroblasts) cell types.CD40L is expressed primarily on activated T cells. B cells, andplatelets. Much of the understanding of CD40/CD40L biology comes fromthe interaction between APCs (CD40 expression on either dendritic cells(DC) or B cells) and CD40L-expressing T cells. On resting B cells. CD40Lengagement drives B cell activation, proliferation, and memory B celldevelopment (Kehry, Immunol. 1996 Apr. 1; 156(7):2345-8). CD40 signalingis also required for immunoglobulin class switching and germinal centerformation. The importance of the CD40/CD40L signaling pathway in B cellbiology is evident in CD40- or CD40L-deficient mice which lack germinalcenters and T-dependent antibody responses are suppressed. However,T-independent IgG responses remain intact in CD40−/− mice suggestingthat it is cell-cell interaction that is lacking in these mice.CD40-deficient mice also have deficits in the T cell compartment.Signaling through CD40 on dendritic cells upregulates MHC class II aswell as various costimulatory molecules such as CD80 and CD86 andpromotes maturation of DC. Mature DC stimulate activation and survivalof CD4+ T cells through production of cytokines such as IL-2 and IL-12.Inefficient T cell priming appears to be the primary cause ofcompromised T-dependent humoral responses in CD40L−/− mice (Grewal,Nature, 1995 Dec. 7; 378(6557):617-20). A similar B cell phenotype canbe seen in humans with X-linked hyper IgM syndrome. These patientssuffer from primary immunodeficiency due to mutations in the CD40L locusthat abrogates CD40/CD40L signaling. These individuals have elevated IgMlevels and cannot produce IgA, IgG, and IgE resulting in an increasedrisk of opportunistic infections (Adriana, J Clin Immunol. 2008 May; 28Suppl 1:S62-6).

CD40 signaling pathway is central to the conversion of resting or naïvelymphocytes and APCs to an activated/mature phenotype. Although T cellpriming and B cell activation can occur in the absence of CD40/CD40Lsignaling, this pathway is required for generating a robust adaptiveimmune response. Engagement of CD40 by CD40L results in the recruitmentof TNF receptor associated factors (TRAFs) to the cytoplasmic domain ofCD40 (Bishop. Adv Exp Med Biol. 2007; 597:131-51). Phosphorylation ofvarious TRAF proteins results in activation of both canonical andnon-canonical NFkB pathways. In addition, JAK3 association with CD40cytoplasmic tail results in STAT5 activation which induces maturation ofDC as well as TNF and IFNγ production. TRAF6-dependent PI3K activationis a critical survival signal in DC while TRAF2/TRAF6 have redundantfunctions in NFkB activation and upregulation of CD80 expression(Hostager, J Biol Chem. 2003 Nov. 14; 278(46):45382-90). TRAFs 2, 3, 5,and 6 have all been shown to play an important role in immunoglobulinclass switching mediated by CD40 signaling (Leo, Proc Natl Acad Sci USA.1999 Feb. 16; 96(4): 1421-1426).

CD40/CD40L signaling pathway has been implicated in the pathogenesis ofmany autoimmune diseases including systemic lupus erythematosus (SLE),inflammatory bowel disease (IBD), multiple sclerosis, rheumatoidarthritis, and Sjogren's syndrome (Law and Grewal. Adv Exp Med Biol.2009; 647:8-36). CD40 expression is elevated on macrophages,endothelium, epithelium, and B cells in tissues damaged by chronicautoimmunity including kidney, intestine, and joints (Borcherding, Am JPathol. 2010 April; 176(4): 1816-27; Sawada-Hasc, Am J Gastroenterol.2000 June; 95(6): 1516-23). Soluble CD40L is elevated in patientssuffering from SLE, IBD, and Sjogren's syndrome consistent withinflammatory burden in these patients.

Some of the earliest evidence the CD40/CD40L pathway in chronicintestinal inflammation came from preclinical models where anti-CD40LmAbs protected rodents from experimental colitis (de Jong,Gastroenterology. 2000 September; 119(3):715-23; Liu, J Immunol. 2000Jun. 1; 164(11):6005-14: Stuber, J Exp Med 1996 Feb. 1, 183(2):693-8).Reduction in disease activity scores were associated with reducedpro-inflammatory cytokine production in the gut and protection fromchronic body weight loss. Similar results were observed in animals thatwere genetically deficient for CD40 or CD40L (de Jong, Gastroenterology.2000 September; 119(3):715-23). Treatment of mice with anti-CD40L mAbsafter disease onset is still effective in reducing disease activitysuggesting that this pathway is critical for maintenance of chronicinflammatory disease. In addition, CD40 agonist antibodies aresufficient to drive intestinal inflammation in mice that lacklymphocytes (Uhlig, Immunity. 2006 August; 25(2):309-18). More recentdata using CD40 siRNA also point to an important role for CD40 signalingin colitis (Arranz, J Control Release. 2013 Feb. 10; 165(3):163-72). InCrohn's disease, lamina propria monocytes and epithelium express highlevels of CD40 and CD40+ monocytes are enriched in peripheral blood.Furthermore, polymorphisms in the CD40 locus have been linked toincreased susceptibility to IBD. In Crohn's patients treated withanti-TNF antibodies, transcriptional profiling indicates that CD40 mRNAlevels decrease in patients with an adequate drug treatment response.However, in patients with a poor response to TNF inhibitors. CD40 mRNAlevels are unchanged suggesting that CD40-dependent, TNF-independentpathways may promote inflammation in these patients. Studies suggestthat inhibition of CD40 mediated signaling is important in thepathogenesis of IBD as well as other autoimmune diseases. Accordingly,there remains a need for antagonist anti-CD40 antibodies, andantigen-binding portions thereof, that can be used for therapeuticpurposes for treating chronic inflammatory diseases and disorders, suchCrohn's disease.

SUMMARY OF THE INVENTION

This invention pertains to antagonist anti-CD40 antibodies, orantigen-binding portions thereof. Antibodies of the inventions include,but are not limited to, antagonist humanized antibodies, andantigen-binding portions thereof, that are capable of binding human CD40and are substantially free of agonist activity.

In a first aspect, the present invention features an isolated antibody,or antigen binding portion thereof, wherein the antibody, or antigenbinding fragment thereof, binds an epitope of human CD40 defined by thetopographic regions Cys62-Phe67, Gln79-Cys83, Arg90-Thr99, andThr24-Cys37 of SEQ ID NO: 1. In one embodiment, the antibody, or antigenbinding portion thereof, is an antagonist antibody. In one embodiment,the antibody, or antigen binding portion thereof, is an antagonistantibody which is substantially free of agonist activity.

In another embodiment, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR3 having theamino acid sequence of SEQ ID NO: 8 and a light chain variable regioncomprising a CDR3 having the amino acid sequence of SEQ ID NO: 12. In afurther embodiment, the antibody, or antigen binding portion thereof,comprises a heavy chain variable region comprising a CDR2 having theamino acid sequence of SEQ ID NO: 111 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 11. Inanother further embodiment, the antibody, or antigen binding portionthereof, comprises a heavy chain variable region comprising a CDR2having the amino acid sequence of SEQ ID NO: 42 and a light chainvariable region comprising a CDR2 having the amino acid sequence of SEQID NO: 11. In another embodiment, the antibody, or antigen bindingportion thereof, comprises a heavy chain variable region comprising aCDR1 having the amino acid sequence of SEQ ID NO: 6 and a light chainvariable region comprising a CDR1 having the amino acid sequence of SEQID NO: 21.

In one embodiment, the antibody, or antigen binding portion thereof, isan IgG isotype.

In a further related embodiment, the antibody, or antigen bindingportion thereof, is an IgG1 or an IgG4 isotype.

In one embodiment, the antibody, or antigen binding portion thereof, hasan IC50 of at least 50 nM in a Jurkat cell reporter assay.

In another aspect, the present invention features an antagonistanti-CD40 antibody, or antigen-binding portion thereof, comprising alight chain variable region comprising a CDR3 having an amino acidsequence as set forth in SEQ ID NO: 12 and/or a heavy chain variableregion comprising a CDR3 having an amino acid sequence as set forth inSEQ ID NO: 8. In one embodiment, the light chain variable region of theantagonistic anti-CD40 antibody, or antigen-binding portion thereof,comprises a CDR3 having the amino acid sequence as set forth in SEQ IDNO: 12 and wherein the heavy chain variable region comprises a CDR3having the amino acid sequence as set forth in SEQ ID NO: 8. In anotherembodiment, the heavy chain variable region of the antagonist anti-CD40antibody, or antigen-binding portion thereof, further comprises a CDR2having an amino acid sequence as set forth in SEQ ID NO: 42. In anotherfurther embodiment, the light chain variable region of the antagonistanti-CD4 antibody, or antigen-binding portion thereof, further comprisesa CDR2 having an amino acid sequence set forth in SEQ ID NO: 11. Inanother embodiment, the heavy chain variable region of the antagonistanti-CD40 antibody, or antigen-binding portion thereof, furthercomprises a CDR1 having an amino acid sequence as set forth in SEQ IDNO:6. In another further embodiment, the light chain variable region ofthe antagonist anti-CD40 antibody, or antigen-binding portion thereof,further comprises a CDR1 having an amino acid sequence as set forth inSEQ ID NO: 21.

In another embodiment, the antagonistic anti-CD40 antibody, orantigen-binding portion thereof, comprises a heavy chain variable regioncomprising a CDR set of SEQ ID NOs: 6, 42, and 8, and a light chainvariable region comprising a CDR set of SEQ ID NOs: 21, 11, and 12.

In one embodiment, the antagonist anti-CD40 antibody, or antigen bindingportion thereof, is humanized. In a further embodiment, the antagonistanti-CD40 antibody, or antigen binding portion thereof, furthercomprises a human acceptor framework. In a further related embodiment,the human acceptor framework comprises an amino acid sequence selectedfrom SEQ ID NOs: 82-106. In another embodiment, the human acceptorframework comprises at least one framework region amino acidsubstitution, wherein the amino acid sequence of the framework is atleast 65% identical to the sequence of said human acceptor framework andcomprises at least 70 amino acid residues identical to said humanacceptor framework. In a further embodiment, the human acceptorframework comprises at least one framework region amino acidsubstitution at a key residue, said key residue selected from:

a residue adjacent to a CDR;

a glycosylation site residue;

a rare residue;

a residue capable of interacting with human CD40:

a residue capable of interacting with a CDR;

a canonical residue;

a contact residue between heavy chain variable region and light chainvariable region; a residue within a Vernier zone; and

a residue in a region that overlaps between a Chothia-defined variableheavy chain CDR1 and a Kabat-defined first heavy chain framework.

In a further related embodiment, the key residue is selected from 48H,49H, and 36L. In one embodiment, the key residue substitution is in thevariable heavy chain region and is V48I or S49A. In another embodiment,the key residue substitution is in the variable light chain region andis Y36F.

In one embodiment, the antagonist anti-CD40 antibody, or antigen bindingportion thereof, comprises a heavy chain variable region comprising theamino acid sequence set forth in SEQ ID NO: 28. In another embodiment,the antagonist anti-CD40 antibody, or antigen binding portion thereof,comprises a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO: 20.

In one embodiment, the antagonist anti-CD40 antibody, or antigen bindingportion thereof, is substantially free of agonist activity. In anotherembodiment, the antagonist anti-CD40 antibody, or antigen-bindingportion thereof, inhibits the binding of CD40 to CD40 ligand (CD40L) orto soluble CD40 ligand (sCD40L). In another further embodiment, theantagonist anti-CD40 antibody, or antigen-binding portion thereof, bindscyno CD40. In one embodiment, the anti-CD40 antibody, or antigen-bindingportion thereof, binds human and cyno CD40, but does not bind rat,rabbit, or mouse CD40.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, is capable of modulating a biologicalfunction of CD40. In a further embodiment, the antagonist anti-CD40antibody, or antigen-binding portion thereof, is capable of neutralizingCD40. In still another further embodiment, the antagonist anti-CD40antibody, or antigen-binding portion thereof, inhibits NF-κB activation.

In one embodiment, the antagonist anti-CD40 antibody, or antigen-bindingportion thereof, has an on rate constant (K_(on)) to CD40 selected fromat least about 10²M⁻¹s⁻¹; at least about 10³M⁻¹s⁻¹; at least about10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; and at least about 10⁶M⁻¹s⁻¹; asmeasured by surface plasmon resonance.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, has a dissociation constant (K_(D)) toCD40 selected from the group consisting of: at most about 10⁻⁷ M; atmost about 10⁻⁸ M; at most about 10⁻⁹ M; at most about 10⁻¹⁰ M; at mostabout 10⁻¹¹ M; at most about 10⁻¹² M; and at most 10⁻¹³ M.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, comprises a heavy chain immunoglobulinconstant domain of a human IgM constant domain, a human IgG1 constantdomain, a human IgG2 constant domain, a human IgG3 constant domain, ahuman IgG4 constant domain, a human IgA constant domain, or a human IgEconstant domain. In a related embodiment, the heavy chain immunoglobulinconstant region domain of the antagonist anti-CD40 antibody, orantigen-binding portion thereof, is a human IgG1 constant domain. In afurther related embodiment, the human IgG1 constant domain of theantagonist anti-CD40 antibody, or antigen-binding portion thereof,comprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, further comprises a light chainimmunoglobulin constant domain comprising a human Ig kappa constantdomain or a human Ig lambda constant domain. In a related embodiment,the human Ig kappa constant domain of the antagonist anti-CD40 antibody,or antigen-binding portion thereof, comprises an amino acid sequence ofSEQ ID NO:4 or wherein the human Ig lambda constant domain comprises anamino acid sequence SEQ ID NO:81.

The present invention also features, in certain embodiments, anantagonist anti-CD40 antibody, or antigen-binding portion thereof, thatcompetes with the antibody, or antigen binding portion thereof, as setforth in any of the aspects and embodiments described herein.

In another aspect, the present invention features an antagonistanti-CD40 antibody, or antigen-binding portion thereof, that comprises aheavy chain CDR1 comprising an amino acid sequence as set forth in SEQID NO:6, a heavy chain CDR2 comprising an amino acid sequence as setforth in SEQ ID NO:42, a heavy chain CDR3 comprising an amino acidsequence as set forth in SEQ ID NO:8, a light chain CDR1 comprising anamino acid sequence as set forth in SEQ ID NO:21, a light chain CDR2comprising an amino acid sequence as set forth in SEQ ID NO: 11, and alight chain CDR3 comprising an amino acid sequence as set forth in SEQID NO:12.

In another aspect, the present invention features an antagonistanti-CD40 antibody, or antigen-binding portion thereof, comprising aheavy chain variable domain comprising an amino acid sequence set forthin SEQ ID NO: 28 and a light chain variable domain comprising an aminoacid sequence set forth in SEQ ID NO: 20. In another aspect, the presentinvention features an antagonist anti-CD40 antibody, or antigen-bindingportion thereof, comprising a heavy chain variable domain comprising anamino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 28, and/or a light chain variable domaincomprising an amino acid sequence having at least 90%, 95%, 96%, 97%,98%, or 99% identity to SEQ ID NO: 20.

In another aspect, the present invention features an antagonistanti-CD40 antibody, or antigen-binding portion thereof, comprising aheavy chain comprising an amino acid sequence set forth in SEQ ID NO:41, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 41, and/or a light chain comprising an amino acidsequence set forth in SEQ ID NO: 40, or a sequence having at least 90%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 40. In one embodiment,the heavy chain of the antagonist anti-CD40 antibody, or antigen bindingportion thereof, comprises an amino acid sequence set forth in SEQ IDNO: 41, and the light chain of the antagonistic anti-CD40 antibody, orantigen binding portion thereof, comprises an amino acid sequence setforth in SEQ ID NO: 40.

In another aspect, the present invention features an anti-CD40 antibodycomprising a heavy chain comprising an amino acid sequence as set forthin SEQ ID NO: 41, and a light chain comprising an amino acid sequence asset forth in SEQ ID NO: 40.

In one embodiment, the antibodies, or antigen binding portions thereof,of the invention are recombinant.

The present invention also features, in certain embodiments, apharmaceutical composition comprising the anti-CD40 antibody, or antigenbinding portion thereof, as set forth in any of the aspects andembodiments described herein, and a pharmaceutically acceptable carrier.

The present invention also features, in other certain embodiments, apharmaceutical composition comprising the anti-CD40 antibody, or antigenbinding portion thereof, as set forth in any of the aspects andembodiments described herein, and a polysorbate. In a further relatedembodiment, the polysorbate is polysorbate 80.

In another embodiment, the pharmaceutical composition comprises ahistidine buffer.

In another further embodiment, the pharmaceutical composition comprisesa polyol. In a related embodiment, the polyol is selected from mannitol,sorbitol, trehalose, or sucrose.

In another embodiment, the pharmaceutical composition has a pH of about4 to about 8. In a related embodiment, the pharmaceutical compositionhas a pH of about 5 to about 7.

In another embodiment, the pharmaceutical composition is lyophilized.

The present invention also features, in other embodiments, an isolatednucleic acid encoding an antagonist anti-CD40 antibody amino acidsequence of any one of the aspects and embodiments described herein. Ina further embodiment, the present invention features a vector comprisingthe isolated nucleic acid. In a related embodiment, the vector isselected from pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ vectors.

In another embodiment, a host cell comprises the vector. In a relatedembodiment, the host cell is a prokaryotic cell or a eukaryotic cell. Ina further embodiment, the eukaryotic cell is a protist cell, an animalcell, a plant cell, a fungal cell, a yeast cell, a mammalian cell, anavian cell, or an insect cell. In another further embodiment, themammalian cell is a CHO cell or a COS cell.

The present invention also features, in certain embodiments, a method ofproducing an antagonist anti-CD40 antibody, or antigen binding portionthereof, the method comprising the steps of culturing a host cell of anyone of the aspects and embodiments described herein in culture mediumunder conditions sufficient to produce the antagonist anti-CD40antibody, or antigen binding portion thereof. In further embodiments, anantagonist anti-CD40 antibody, or antigen binding portion thereof, isproduced by the method.

The present invention also features, in other embodiments, a method forreducing human CD40 activity, the method comprising the step ofcontacting human CD40 with the antibody, or antigen-binding portionthereof, of any one of the aspects and embodiments described herein,such that human CD40 activity is reduced. In a further embodiment, themethod is an in vitro method.

The present invention also features, in other certain embodiments, amethod for treating a human subject having a disorder in which CD40 isdetrimental comprising administering an effective amount of theanti-CD40 antibody, or antigen binding portion thereof, of any one ofthe aspects and embodiments described herein, to the subject.

The present invention also features, in other embodiments, a method forreducing human CD40 activity in a human subject having a disorder inwhich CD40 activity is detrimental, the method comprising the step ofadministering to the human subject the antibody, or antigen bindingportion thereof, of any one of aspects and embodiments described herein,such that human CD40 activity in the human subject is reduced.

In a further embodiment, the antibody, or antigen binding portionthereof, is administered before, concurrently, or after theadministration of a second agent to the subject. In a further relatedembodiment, the second agent is selected from an antibody, or fragmentthereof, capable of binding human IL-12; PGE2; LPA; NGF; CGRP; SubP;RAGE; histamine; a histamine receptor blocker; bradykinin; IL-1 alpha;IL-1beta; VEGF; PLGF; methotrexate; a corticosteroid, a glucocorticoidreceptor modulator; cyclosporin, rapamycin, FK506, a non-steroidalanti-inflammatory agent, an inhaled steroid; beta-agonist; short-actingor long-acting beta-agonist; antagonist of leukotrienes or leukotrienereceptors; ADVAIR; IgE inhibitor; anti-IgE antibodies; XOLAIR;phosphodiesterase inhibitor; PDE4 inhibitor; xanthine; anticholinergicdrug; mast cell-stabilizing agent; Cromolyn: IL-4 inhibitor; IL-5inhibitor; eotaxin/CCR3 inhibitors antagonists of histamine or itsreceptors including H1, H2, H3, and H4; antagonists of prostaglandin Dor its receptors DP1 and CRTH2; TNF antagonist: a soluble fragment of aTNF receptor, ENBREL; TNF enzyme antagonist: TNF converting enzyme(TACE) inhibitor: muscarinic receptor antagonist: TGF-beta antagonist;interferon gamma: perfenidone; chemotherapeutic agent, methotrexate;leflunomide; sirolimus (rapamycin) or an analog thereof. CCI-779; COX2or cPLA2 inhibitor; NSAID; immunomodulator; p38 inhibitor; TPL-2, MK-2and NFkB inhibitor; budenoside: epidermal growth factor; corticosteroid:cyclosporine; sulfasalazine; aminosalicylate; 6-mercaptopurine:azathioprine; metronidazole; lipoxygenase inhibitor; mesalamine;olsalazine; balsalazide; antioxidant; thromboxane inhibitor; IL-1receptor antagonist; anti-IL-1β antibody; anti-IL-6 antibody; growthfactor; elastase inhibitor; pyridinyl-imidazole compound; antibody oragonist of LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF, or PDGF; antibody of CD2,CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligand;FK506: rapamycin; mycophenolate mofetil; ibuprofen; prednisolone;phosphodiesterase inhibitor; adensosine agonist; antithrombotic agent;complement inhibitor; adrenergic agent; IRAK, NIK, IKK, p38, or MAPkinase inhibitor; IL-1β converting enzyme inhibitor: TNF-α.quadrature,converting enzyme inhibitor; T-cell signaling inhibitor;metalloproteinase inhibitor, 6-mercaptopurine; angiotensin convertingenzyme inhibitor; soluble cytokine receptor; soluble p55 TNF receptor,soluble p75 TNF receptor, sIL-1RI; sIL-1RII; sIL-6R; anti-inflammatorycytokine; IL-4; IL-10; IL-11; or TGF-β.

In a further embodiment, the disorder is selected from a respiratorydisorder; asthma; allergic and nonallergic asthma; asthma due toinfection; asthma due to infection with respiratory syncytial virus(RSV); chronic obstructive pulmonary disease (COPD): a conditioninvolving airway inflammation; eosinophilia; fibrosis and excess mucusproduction; cystic fibrosis; pulmonary fibrosis; an atopic disorder;atopic dermatitis; urticaria; eczema; allergic rhinitis; allergicenterogastritis; an inflammatory and/or autoimmune condition of theskin; an inflammatory and/or autoimmune condition of gastrointestinalorgans; inflammatory bowel diseases (IBD); ulcerative colitis; Crohn'sdisease; an inflammatory and/or autoimmune condition of the liver: livercirrhosis: liver fibrosis; liver fibrosis caused by hepatitis B and/or Cvirus; scleroderma; tumors or cancers; hepatocellular carcinoma;glioblastoma; lymphoma; Hodgkin's lymphoma; a viral infection; abacterial infection; a parasitic infection; HTLV-1 infection;suppression of expression of protective type 1 immune responses, andsuppression of expression of a protective type 1 immune response duringvaccination.

In another further embodiment, the disorder is selected from anautoimmune or inflammatory disease, such as systemic lupus erythematosus(SLE), discoid lupus, lupus nephritis, sarcoidosis, inflammatoryarthritis, including, but not limited to, juvenile arthritis, rheumatoidarthritis, psoriatic arthritis, Reiter's syndrome, ankylosingspondylitis, and gouty arthritis, rejection of an organ or tissuetransplant, hyperacute, acute, or chronic rejection and/or graft versushost disease, multiple sclerosis, hyper IgE syndrome, polyarteritisnodosa, primary biliary cirrhosis, inflammatory bowel disease. Crohn'sdisease, celiac's disease (gluten-sensitive enteropathy), autoimmunehepatitis, pernicious anemia, autoimmune hemolytic anemia, psoriasis,scleroderma, myasthenia gravis, autoimmune thrombocytopenic purpura,autoimmune thyroiditis, Grave's disease, Hasimoto's thyroiditis, immunecomplex disease, chronic fatigue immune dysfunction syndrome (CFIDS),polymyositis and dermatomyositis, cryoglobulinemia, thrombolysis,cardiomyopathy, pemphigus vulgaris, pulmonary interstitial fibrosis,sarcoidosis, Type I and Type II diabetes mellitus, type 1, 2, 3, and 4delayed-type hypersensitivity, allergy or allergic disorders,unwanted/unintended immune responses to therapeutic proteins, asthma,Churg-Strauss syndrome (allergic granulomatosis), atopic dermatitis,allergic and irritant contact dermatitis, urtecaria, IgE-mediatedallergy, atherosclerosis, vasculitis, idiopathic inflammatorymyopathies, hemolytic disease, Alzheimer's disease, chronic inflammatorydemyelinating polyneuropathy, Sjogren's, and psoriasis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat an inflammatorybowel disease (IBD).

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat ulcerativecolitis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat Crohn's disease.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat systemic lupuserythematosus (SLE).

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat sarcoidosis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat juvenilearthritis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat rheumatoidarthritis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat psoriaticarthritis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat ankylosingspondylitis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat hidradenitissuppurativa.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat uveitis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention. e.g. Ab102, is used to treat Sjogren's.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention. e.g. Ab102, is used to treat psoriasis.

In another further embodiment, the antibody, or antigen binding fragmentthereof, is administered by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intra-articular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracerebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal,and transdermal.

The present invention also features, in other certain embodiments, amethod of determining the presence of CD40 or fragment thereof in a testsample by an immunoassay, wherein the immunoassay comprises contactingthe test sample with at least one antibody, or antigen-binding portionthereof, of any of the aspects and embodiments described herein, and atleast one detectable label. In a further embodiment, the method furthercomprises the steps of: (i) contacting the test sample with the at leastone antibody, or antigen-binding portion thereof, wherein the antibody,or antigen-binding portion thereof, binds to an epitope on the CD40 orfragment thereof so as to form a first complex; (ii) contacting thecomplex with the at least one detectable label, wherein the detectablelabel binds to an epitope on the first complex, or on the CD40 orfragment thereof, that is not bound by the antibody, or antigen-bindingportion thereof, to form a second complex; and (iii) detecting thepresence of the CD40 or fragment thereof in the test sample based on thesignal generated by the detectable label in the second complex, whereinthe presence of the CD40 or fragment thereof is directly correlated withthe signal generated by the detectable label. In a further relatedembodiment, the method further comprises the steps of: (i) contactingthe test sample with the at least one antibody, or antigen-bindingportion thereof, wherein the antibody, or antigen-binding portionthereof, binds to an epitope on the CD40 or fragment thereof so as toform a first complex; (ii) contacting the complex with the at least onedetectable label, wherein the detectable label competes with the CD40 orfragment thereof for binding to the antibody, or antigen-binding portionthereof, so as to form a second complex; and (iii) detecting thepresence of the CD40 or fragment thereof in the test sample based on thesignal generated by the detectable label in the second complex, whereinthe presence of the CD40 or fragment thereof is indirectly correlatedwith the signal generated by the detectable label.

In one embodiment, the invention provides a DVD-Ig which comprises thebinding regions, e.g. CDRs, described herein. In one embodiment, theDVD-Ig of the invention comprises four polypeptide chains, wherein twopolypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is afirst heavy chain variable domain, VD2 is a second heavy chain variabledomain, C is a heavy chain constant domain, X1 is a linker with theproviso that it is not CH1, and X2 is an Fc region; and two polypeptidechains comprise VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first lightchain variable domain, VD2 is a second light chain variable domain, C isa light chain constant domain. X1 is a linker with the proviso that itis not CH1, and X2 does not comprise an Fc region; and n is 0 or 1;wherein said four polypeptide chains of said binding protein form fourfunctional antigen binding sites. In one embodiment, the first (and/orsecond) heavy chain of the DVD comprises a CDR set as set forth in SEQID NOs: 6, 42, and 8. In one embodiment, the first (and/or second) lightchain variable region comprises a CDR set as set forth in SEQ ID NOs:21, 11, and 12. In one embodiment, the DVD-Ig of the invention ismonospecific and binds huCD40. In another embodiment, the DVD-Ig of theinvention is multispecific and binds CD40 and a second molecular target.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A graphically depicts the antagonistic activity of chimericantibody (Antibody 1 (Ab1)) versus an agonist control and knownantagonist antibodies (4D11 (Astellas) and Bib (Boehringer)). FIG. 1Bgraphically depicts the activity of the same chimeric antibody Ab1 in anagonist assay using the same agonist and antagonist controls as FIG. 1A.

FIGS. 2A and 2B graphically depict agonist activity (FIG. 2A) andantagonist activity (FIG. 2B) of humanized antibody Ab101 in comparisonto antibody 4D11, antibody Bib, an IgG antibody (control), and anagonist control antibody (2141).

FIGS. 3A and 3B graphically depict results from in vivo studies ofhumanized antibody Ab101. FIG. 3A graphically depicts IgG production inhuscid mice who have received human PBMCs in combination with an Igcontrol, a CTLA4-Ig fusion, or the Ab101 antibody. FIG. 3B graphicallydepicts B cell survival in the same mouse model administered the sameagents as FIG. 3A.

FIG. 4A and FIG. 4B show an amino acid sequence alignment of anti-humanCD40 murine antibody antagonists, and the alignment consensus sequences.FIG. 4A shows a sequence alignment of the variable light chains ofAntibody 3 (Ab3) (SEQ ID NO:48), Ab1 (SEQ ID NO:9) and Antibody 2 (Ab2)(SEQ ID NO: 76) and the variable light chain consensus sequence (SEQ IDNO: 116). FIG. 4B shows a sequence alignment of the variable heavychains of Ab3 (SEQ ID NO:44). Ab1 (SEQ ID NO:5) and Ab2 (SEQ ID NO:75)and the variable heavy chain consensus sequence (SEQ ID NO: 117).

FIGS. 5A and 5B graphically depict representative neutralization potency(antagonist activity) (FIG. 5A) and agonist activity (FIG. 5B) of Ab102on human CD40 in the monocyte activation assays described in Example 7.Monoctyte activation corresponds with increases in TNF concentrationwithin each assay.

FIG. 6 graphically depicts the dose responsive inhibition of endoscopyscore with prophylactic administration of antibody 138 (Ab138). Antibody138 was tested at doses of 15, 5, 1.5 and 0.5 mg/kg. An IgG negativecontrol was used. Anti p40IL-12/23 treatment was used as a positivecontrol. Disease is mediated by CD45Rbhi cells that are transferred toanimals. RBlow refers to the negative control group. CD45RBlow cells donot mediate disease.

FIG. 7 graphically depicts the results of immunohistochemical analysisto determine IBA1+ macrophages in the colon with administration ofantibody 138 (Ab138). Histological analysis of colonic sections showed adecrease in macrophages (general measure of inflammation). An IgGnegative control was used. Anti p40IL-12/23 treatment was used as apositive control.

FIG. 8 graphically depicts serum levels of circulating antibody 138(Ab138) 96 hours (equal to C_(trough)) after the final dose in theT-cell transfer model of colitis. The serum levels were shown to be doseresponsive. Anti p40IL-12/23 treatment was used as a positive control.Only 1 animal in the 0.5 mg/kg group has measurable levels of Ab138.

FIG. 9A graphically depicts endoscopy results following administrationof antibody 138 in a colitis mouse model. Antibody 138 (Ab138) treatmentwas initiated three weeks post cell injection, following confirmation ofendoscopic disease, and a dose responsive inhibition of the MEDAI sumscore was noted. The highest dose (15 mg/kg) reached statisticalsignificance (FIG. 9A).

FIG. 9B graphically depicts histology results following administrationof antibody 138. Histological analysis of IBA1+ macrophages in the colonas a measure of myeloid inflammation is shown in FIG. 9B.

FIG. 10 graphically depicts results showing that Ab102 suppressed antiKLH IgM and anti KLH IgG (dashed line) as compared to control animalstreated with vehicle only (solid lines). Cynomolgus monkeys(two/sex/group) were administered Ab102 at dosages of 0 (vehicle only)or 10 mg/kg subcutaneously (SC) for 5 weeks. Keyhole limpet hemocyanin(KLH) was administered to all animals on Day 8. Serum samples werecollected from each animal at −11, −7, 0, 4, 7, 10, 14 and 21 daysrelative to KLH administration (KLH days).

FIG. 11A is a graph that shows anti-CD40 antibody 138 treatmentprevented proteinuria in MRL/lpr mice. Mice were dosed with 15 mg/kg ofantibody 2×/week, 5 mg/kg of antibody 2×/week, 1.5 mg.kg of antibody2×/week or 15 mg/kg antibody 1×/week. Administration of phosphatebuffered saline (PBS) vehicle alone was used as a control. Proteinuriawas determined as percent urine protein <300 mg/dL.

FIG. 11B is a graph that depicts results showing that anti-CD40 antibody138 treatment extended survival of MRL/lpr mice. Animals were dosed with15 mg/kg of antibody 2×/week, 5 mg/kg of antibody 2×/week, 1.5 mg.kg ofantibody 2×/week or 15 mg/kg antibody 1×/week. Administration of vehiclealone was used as a control. Percent survival was indicated over time.

FIG. 12A is a graph that depicts results showing that anti-CD40 antibody138 treatment prevented the development of nephritis. FIG. 12A shows theeffect of antibody 138 on glomerular disease in mice dosed with 15 mg/kgof antibody 2×/week. 5 mg/kg of antibody 2×/week, 1.5 mg.kg of antibody2×/week or 15 mg/kg antibody 1×/week, at day 29 and day 63.Administration of PBS vehicle alone was used as a control. Glomerulardisease was assessed on a scale of 0-4. As glomerular disease severityworsened in aging MRL mice, antibody 138 maintained efficacy atminimizing glomerular disease at 5 and 15 mg/kg. Perivascularinflammation was scored on a scale 0-4 based on the following criteria:0—up to a few rare lymphocytes; 1—a few lymphocytes forming looseaggregates; 2—lymphocytes forming discrete small aggregates; 3—polarizedaggregate of lymphocytes that bulge into the lumen of the adjacent veinbut fail to fully surround the arcuate artery; 4—lymphocyte aggregatefully surrounding and extending into the adventitia of the arcuateartery.

FIG. 12B is a graph that depicts results showing that anti-CD40 antibodytreatment prevented the development of nephritis. FIG. 12B depictsresults showing the effect of antibody 138 on kidney perivascular (PV)inflammation in mice dosed with 15 mg/kg of antibody 2×/week. 5 mg/kg ofantibody 2×/week, 1.5 mg.kg of antibody 2×/week or 15 mg/kg antibody1×/week, at day 29 and day 63. Administration of PBS vehicle alone wasused as a control. Anti-CD40 antibody at 5 and 15 mg/kg was effective atreducing perivascular (PV) infiltrates in the kidney at 29 and 63 days.

FIG. 12C is a graph that shows anti-C D40 antibody 138 treatmentprevented the development of nephritis. FIG. 12C shows the effect ofantibody 138 on tubulointerstitial inflammation (TI) in mice dosed with15 mg/kg of antibody 2×/week, 5 mg/kg of antibody 2×/week, 1.5 mg.kg ofantibody 2×/week or 15 mg/kg antibody 1×/week, at day 29 and day 60.Administration of PBS vehicle alone was used as a control. TI wasreduced early in disease.

FIG. 13A is a graph that shows anti-CD40 antibody 138 treatmentprevented salivary gland inflammation. FIG. 13A shows the effect ofantibody 138 on salivary gland inflammation in mice dosed with 15 mg/kgof antibody 2×/week, 5 mg/kg of antibody 2×/week. 1.5 mg.kg of antibody2×/week or 15 mg/kg antibody 1×/week, at day 29 and day 60.Administration of PBS vehicle alone was used as a control. Periductularinflammation was scored on a scale 0-4 based on the following criteria:0—up to a few rare leukocytes; 1—a few leukocytes forming looseaggregates; 2—leukocytes forming discrete small aggregates: 3—polarizedaggregate of leukocytes that fully surround the duct; 4—leukocytesaggregate extending into the glandular parenchyma of the salivary gland.

FIG. 13B is a graph that shows anti-CD40 antibody 138 treatmentprevented joint inflammation. FIG. 13B shows the effect of antibody 138on joint inflammation in mice dosed with 15 mg/kg of antibody 2×/week, 5mg/kg of antibody 2×/week, 1.5 mg.kg of antibody 2×/week or 15 mg/kgantibody 1×/week, at day 29 and day 60. Administration of PBS vehiclealone was used as a control. Joint inflammation was scored for each oftwo paws per mouse on a scale of 0-4 based on the following criteria:0—no inflammation; 1—a few leukocytes in joint space: 2—frequentleukocytes within joint space with mild synovial proliferation;3—leukocytes expanding joint spaces with moderate synovialproliferation; 4—leukocytes and synovial proliferation extending andcoalescing within all joint spaces with marked bone erosion and/orproliferation. The scores were added for a total possible score of 8 permouse.

FIG. 14 is a panel of four graphs (i-iv) that shows that anti-CD40antibody 138 prevented the expansion of follicular helper T cells (Tfh)and germinal center (GC) B cells in the spleen, as determined by flowcytometry. Mice were dosed with 15 mg/kg of antibody 2×/week, 5 mg/kg ofantibody 2×/week, 1.5 mg.kg of antibody 2×/week or 15 mg/kg antibody1×/week. Administration of PBS vehicle alone was used as a control.Panel (i) shows the number of Tfh cells in the spleen at day 29. Panel(ii) shows the number of Tfh cells in the spleen at day 63. Panel (iii)shows the number of GC B cells in the spleen at day 29. Panel (iv) showsthe number of GC B cells in the spleen at day 63.

FIG. 15A is a graph that shows anti-CD40 antibody 138 treatmentprevented an increase in total circulating IgG levels at day 29. Micewere dosed with 15 mg/kg of antibody 2×/week. 5 mg/kg of antibody2×/week, 1.5 mg.kg of antibody 2×/week and 15 mg/kg antibody 1×/week.Administration of PBS vehicle alone was used as a control.

FIG. 15B is a graph that shows anti-CD40 antibody 138 treatmentprevented an increase in total circulating IgG levels at day 63. Micewere dosed with 15 mg/kg of antibody 2×/week, 5 mg/kg of antibody2×/week, 1.5 mg.kg of antibody 2×/week and 15 mg/kg antibody 1×/week.Administration of PBS vehicle alone was used as a control.

FIG. 16A is a graph that shows the effect of anti-CD40 antibody 138treatment on anti-double stranded DNA (anti-dsDNA) titers at day 29.Mice were dosed with 15 mg/kg of antibody 2×/week. 5 mg/kg of antibody2×/week, 1.5 mg.kg of antibody 2×/week or 15 mg/kg antibody 1×/week.Administration of PBS vehicle alone was used as a control. At day 29,anti-dsDNA titers were determined.

FIG. 16B is a graph that shows anti-CD40 antibody 138 treatment onanti-double stranded DNA (anti-dsDNA) titers at day 63. Mice were dosedwith 15 mg/kg of antibody 2×/week, 5 mg/kg of antibody 2×/week, 1.5mg.kg of antibody 2×/week or 15 mg/kg antibody 1×/week. Administrationof PBS vehicle alone was used as a control. At day 63, anti-dsDNA titerswere determined.

FIG. 17A is a graph that shows prophylactic dosing of anti-CD40 antibody138 prevented proteinuria. Prophylactic treatment was started in mice at26 weeks of age, and proteinuric mice were excluded from the study. Micewere dosed with 15 mg/kg of antibody 2×/week, 1.5 mg.kg of antibody2×/week or 15 mg/kg antibody 1×/week. Administration of PBS vehiclealone was used as a control. Proteinuria was determined as percent urineprotein <300 mg/dL.

FIG. 17B is a graph that shows prophylactic dosing of anti-CD40 antibody138 extended survival using an SLE mouse model. Prophlyactic treatmentwas started in mice at 26 weeks of age, and proteinuric mice wereexcluded from the study. Mice were dosed with 15 mg/kg of antibody2×/week, 1.5 mg.kg of antibody 2×/week or 15 mg/kg antibody 1×/week.Administration of PBS vehicle alone was used as a control. Percentsurvival was assessed through 36 weeks of age.

FIG. 18A is a graph that shows that mice treated with antibody 138 at adose of 15 mg/kg IP 2×/week, developed low proteinuria over time, asshown by the urine protein grade (mg/dL equivalent). Vehicle PBSadministered IP, 2×/week was used as a control. Prednisolone was givenat a dose of 10 mg/kg orally (PO), once a day (SID). Neither the vehiclePBS untreated control mice nor the prednisolone treated mice developedlow proteinuria. The threshold of proteinuria is indicated as 300 mg/dL.

FIG. 18B is a graph that shows the rate of recovery from proteinuria inmice treated with antibody 138 at a dose of 15 mg/kp IP 2×/week. Basedon the rate of recovery from proteinuria as determined by percent normalurine protein, the average time to recovery of proteinuria was 23+7days. Vehicle PBS administered IP, 2×/week was used as a control.Prednisolone was given at a dose of 10 mg/kg orally (PO), once a day(SID).

FIG. 18C is a graph that shows that mice treated with anti-CD40 antibody138 at a dose of 15 mg/kp IP 2×/week, significantly extended survival,as shown by percent survival. Vehicle PBS administered IP, 2×/week wasused as a control. Prednisolone was given orally (PO), once a day (SID).

FIG. 19A is a graph that shows that saliva production is preserved byprophylactic treatment with antibody 138 at a dose of 15 mg/kp IP2×/week, 1.5 mg/kg 2×/week, 15 mg/kg 1×/week. Vehicle PBS was used as acontrol. Prednisolone was administered at a dose of 10 mg/kg. Salivaproduction in 7 week old NZBWF-1 mice, which are non-diseased youngermice, was used as a further comparison. Amount of saliva (mg) wasdetermined. Saliva production by anti-CD40 treated mice wascomparatively uniform.

FIG. 19B is a graph that shows that saliva volume is preserved byprophylactic treatment with anti-CD40 antibody 138 at a dose of 15 mg/kpIP 2×/week, 1.5 mg/kg 2×/week, 15 mg/kg 1×/week. Vehicle PBS was used asa control. Prednisolone was administered at a dose of 10 mg/kg. Salivavolume/body weight (mg/gm) was determined. Saliva production byanti-CD40 antibody treated mice was significantly greater than inuntreated control mice.

FIG. 20A is a graph that shows that saliva production was preserved bytherapeutic treatment with anti-CD40 antibody 138 at a dose of 15 mg/kg.Prednisolone was administered at a dose of 10 mg/kg. Saliva productionin 11 week old mice was used as a further comparison. Amount of saliva(mg) was determined.

FIG. 20B is a graph that shows that saliva production is preserved bytherapeutic treatment with anti-CD40 antibody 138 at a dose of 15 mg/kg.Prednisolone was administered at a dose of 10 mg/kg. Saliva productionin 11 week old mice was used as a further comparison. Saliva volume/bodyweight (mg/gm) was determined.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to antagonist anti-CD40 antibodies, orantigen-binding portions thereof, and uses thereof. Various aspects ofthe invention relate to antibodies and antibody fragments, andpharmaceutical compositions thereof, as well as nucleic acids,recombinant expression vectors and host cells for making such antibodiesand fragments. Methods of using the antibodies of the invention todetect human CD40, to inhibit human CD40/CD40L activity, either in vitroor in vivo; and to prevent or treat diseases or disorders such aschronic inflammatory disease and Crohn's disease, are also encompassedby the invention.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

That the present invention may be more readily understood, select termsare defined below.

The term “polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art. An example of an isolated polypeptideis an isolated antibody, or antigen-binding portion thereof.

The term “recovering” as used herein, refers to the process of renderinga chemical species such as a polypeptide substantially free of naturallyassociated components by isolation. e.g., using protein purificationtechniques well known in the art.

The terms “human CD40” and “human CD40 wild type” (abbreviated herein ashCD40, hCD40wt), as used herein, refers to a type I transmembraneprotein. In one embodiment, the term human CD40 is intended to includerecombinant human CD40 (rhCD40), which can be prepared by standardrecombinant expression methods. Table 1 provides the amino acid sequenceof human CD40 (i.e., SEQ ID NO. 1), and the extracellular domain thereof(i.e., SEQ ID NO:107), which are known in the art.

TABLE 1 Sequence of human CD40 Sequence Identifier Protein Sequence SEQID NO.: 1 Human CD40 MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDG KESRISVQERQ SEQ ID NO.: 107 HumanCD40 EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTE ExtracellularTECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQ Domain QKGTSETDTICTCEEGWHCTSEACESCV

“Biological activity” as used herein, refers to all inherent biologicalproperties of the CD40 receptor. Biological properties of CD40 includebut are not limited to binding CD40L; involvement in B cell development;involvement in lymphocyte activation; involvement in antigen presentingcells function; regulating activity of dendritic cells, macrophages andB cells; inducing production of inflammatory cytokines in macrophagesand dendritic cells; up-regulating antigen presentation; up-regulating Tcell stimulation; and promoting immunoglobulin class switching in Bcells.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species; for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “agonist”, as used herein, refers to a modulator that, whencontacted with a molecule of interest, e.g., CD40, causes an increase inthe magnitude of a certain activity or function of the molecule comparedto the magnitude of the activity or function observed in the absence ofthe agonist.

The term “antagonist” or “inhibitor”, as used herein, refers to amodulator that, when contacted with a molecule of interest causes adecrease in the magnitude of a certain activity or function of themolecule compared to the magnitude of the activity or function observedin the absence of the antagonist. Particular antagonists of interestinclude those that block or modulate the biological or immunologicalactivity of human CD40 (hCD40). An antagonist antibody of hCD40 may, forexample, inhibit CD86 upregulation of primary human B cells that arecultured with (or exposed to) CD40L (such as culturing the B cells withCD40L-expressing human T cells). In one embodiment, an antagonistanti-CD40 antibody, or antigen-binding portion thereof, that issubstantially free of agonist activity is defined as having a level ofactivity that is equivalent to or within one standard deviation from anegative control in an agonist assay, such as the agonist monocyte assaydescribed in Example 7.

The antibody, or antigen binding portion thereof, of the presentinvention is an antagonist antibody, or antigen binding portion thereof,which causes a decrease in CD40 activity or function as compared to CD40activity or function in the absence of the antibody, or antigen bindingportion thereof. In particular embodiments, the antibody, or antigenbinding portion thereof, is substantially free of agonist activity,i.e., the antibody, or antigen binding portion thereof, does not causean increase in the magnitude of CD40 activity or function as compared toCD40 activity or function in the absence of the antibody, or antigenbinding portion thereof. Agonist and antagonist activity can also beassessed using methods known in the art, e.g., using a CD40 expressingreporter cell line expressing human CD40 linked to NFkB mediatedalkaline phosphatase (AP) or a B cell assay. Further, in one embodiment,agonist and antagonist activity can be assessed using the in vitromonocyte agonist and antagonist assays described in Example 7.

The term “inhibit binding to CD40L” refers to the ability of theantibody, or antigen binding fragment thereof, to prevent the binding ofCD40 to the ligand, CD40L. Such inhibition of binding to CD40L wouldresult in diminishing or abolishing the biological activity mediated bybinding of CD40 to CD40L.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Non-limitingembodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2) or subclass.

The term “antigen binding portion” or “antigen binding fragment” of anantibody (or simply “antibody portion” or “antibody fragment”), as usedherein, refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., hCD40). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Such antibody embodiments mayalso be bispecific, dual specific, or multi-specific formats;specifically binding to two or more different antigens. Examples ofbinding fragments encompassed within the term “antigen-binding portion”or “antigen binding fragment” of an antibody include (i) a Fab fragment,a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii)a F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” or“antigen binding fragment” of an antibody. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger, P., et al.(1993) Proc. Natl. Acad Sci. USA 90:6444-6448; Poljak, R. J., et al.(1994) Structure 2:1121-1123). Such antibody binding portions are knownin the art (Kontermann and Dubel eds., Antibody Engineering (2001)Springer-Verlag, New York. 790 pp. (ISBN 3-540-41354-5).

The term “antibody construct” as used herein refers to a polypeptidecomprising one or more the antigen-binding portions of the inventionlinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen-binding portions.Such linker polypeptides are well known in the art (see e.g., Holliger.P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak. R.J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constantdomain refers to a heavy or light chain constant domain. Human IgG heavychain and light chain constant domain amino acid sequences are known inthe art and represented in Table 2.

TABLE 2 Sequence of human IgG heavy chain constant domain and lightchain constant domain Sequence Sequence Protein Identifier12345678901234567890123456789012 Ig gamma-1 SEQ ID NO.: 2ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY constant regionFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig gamma-1 SEQ ID NO.: 3ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY constant regionFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS mutant LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig Kappa constant SEQ IDNO.: 4 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY reqionPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ig Lambda SEQ ID NO.: 81 QPKAAPSVTLFPPSSEELQANKATLVCLISDFconstant region YPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPSQWKSHRSYSCQVTHEGSTVE KTVAPTECSStill further, an antibody, or antigen-binding portion thereof, may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hCD40 is substantially free of antibodies that specifically bindantigens other than hCD40). An isolated antibody that specifically bindshCD40 may, however, have cross-reactivity to other antigens, such asCD40 molecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The terms “Kabat numbering”, “Kabat definitions and “Kabat labeling” areused interchangeably herein. These terms, which are recognized in theart, refer to a system of numbering amino acid residues which are morevariable (i.e., hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or anantigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci.190:382-391 and, Kabat. E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody or nucleic acid sequence providing or encoding at least80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% ofthe amino acid sequences of one or more of the framework regions. Insome embodiments, the term “acceptor” refers to the antibody amino acidor nucleic acid sequence providing or encoding the constant region(s).In yet another embodiment, the term “acceptor” refers to the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). In a specificembodiment, the term “acceptor” refers to a human antibody amino acid ornucleic acid sequence that provides or encodes at least 80%, preferably,at least 85%, at least 90%/o, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues that does (do) not occur at one or more specific positions of ahuman antibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia et al., J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Bol262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems, although preferred embodiments useKabat or Chothia defined CDRs.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia etal., J. Mol. Biol. 227:799 (1992), both are incorporated herein byreference). According to Chothia et al., critical portions of the CDRsof many antibodies have nearly identical peptide backbone confirmationsdespite great diversity at the level of amino acid sequence. Eachcanonical structure specifies primarily a set of peptide backbonetorsion angles for a contiguous segment of amino acid residues forming aloop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In a preferred embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, CDR-L2,and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain)also divide the framework regions on the light chain and the heavy chaininto four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in whichCDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain acceptor sequences are known in theart. In one embodiment of the invention the human heavy chain and lightchain acceptor sequences are selected from the sequences described inTable 3 and Table 4.

TABLE 3 Heavy Chain Acceptor Sequences SEQ ID Sequence No. Proteinregion 12345678901234567890123456789012 82 VH1-18&JH6 FR1QVQLVQSGAEVKKPGASVKVSCKASGYTFT 83 VH1-18&JH6 FR2 WVRQAPGQGLEWMG 84VH1-18&JH6 FR3 RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR 85 VH1-18&JH6 FR4WGQGTTVTVSS 82 21/28&JH4 FR1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT 86 21/28&JH4FR2 WVRQAPGQRLEWMG 87 21/28&JH4 FR3 RVTITRDTSASTAYMELSSLRSEDTAVYYCAR 8821/28&JH4 FR4 WGQGTLVTVSS 89 VH2-26&JH6 FR1QVTLKESGPVLVKPTETLTLTCTVSGFSLS 90 VH2-26&JH6 FR2 WIRQPPGKALEWLAH 91VH2-26&JH6 FR3 RLTISKDTSKSQVVLTMTNMDPVDTATYYCAR 85 VH2-26&JH6 FR4WGQGTTVTVSS 92 M60&JH4 FR1 QVTLRESGPALVKPTQTLTLTCTLYGFSLS 93 M60&JH4 FR2WIRQPPGKALEWLA 94 M60&JH4 FR3 RLTISKDTSKNQVVLTMTNMDPVDTATYYCAR 88M60&JH4 FR4 WGQGTLVTVSS 82 VH1-46&JH6 FR1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT83 VH1-46&JH6 FR2 WVRQAPGQGLEWMG 95 VH1-46&JH6 FR3RVTMTRDTSTSTVYMSLSSLRSEDTAVYYCAR 85 VH1-46&JH6 FR4 WGQGTTVTVSS

TABLE 4 Light Chain Acceptor Sequences SEQ ID Protein Sequence No.region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

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001)).One of the advantages provided by various embodiments of the presentinvention stems from the recognition that germline antibody genes aremore likely than mature antibody genes to conserve essential amino acidsequence structures characteristic of individuals in the species, henceless likely to be recognized as from a foreign source when usedtherapeutically in that species.

As used herein, the term “key” residues refer to certain residues withinthe variable region that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable region and light chain variableregion, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the term “humanized antibody” is an antibody or avariant, derivative, analog or fragment thereof which immunospecificallybinds to an antigen of interest (e.g., human CD40), and which comprisesa framework (FR) region having substantially the amino acid sequence ofa human antibody and a complementary determining region (CDR) havingsubstantially the amino acid sequence of a non-human antibody. As usedherein, the term “substantially” in the context of a CDR refers to a CDRhaving an amino acid sequence at least 80%, preferably at least 85%, atleast 90%, at least 95%, at least 98% or at least 99% identical to theamino acid sequence of a non-human antibody CDR. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains (Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulin(i.e., donor antibody) and all or substantially all of the frameworkregions are those of a human immunoglobulin consensus sequence.Preferably, a humanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

The humanized antibody can be selected from any class ofimmnunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG1, IgG2, IgG3 and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In a preferred embodiment,such mutations, however, will not be extensive. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

The term “multivalent binding protein” is used in this specification todenote a binding protein comprising two or more antigen binding sites.The multivalent binding protein is preferably engineered to have thethree or more antigen binding sites, and is generally not a naturallyoccurring antibody. The term “multispecific binding protein” refers to abinding protein capable of binding two or more related or unrelatedtargets.

The term “dual variable domain” or “DVD” or “DVD-Ig” as usedinterchangeably herein, are antigen binding proteins that comprise twoor more antigen binding sites and are tetravalent or multivalent bindingproteins. Such DVDs may be monospecific, i.e., capable of binding oneantigen or multispecific, i.e. capable of binding two or more antigens.DVD binding proteins comprising two heavy chain DVD polypeptides and twolight chain DVD polypeptides are referred to a DVD Ig. Each half of aDVD Ig comprises a heavy chain DVD polypeptide, and a light chain DVDpolypeptide, and two antigen binding sites. Each binding site comprisesa heavy chain variable domain and a light chain variable domain with atotal of 6 CDRs involved in antigen binding per antigen binding site. Inone embodiment, the CDRs described herein (e.g., SEQ ID NOs:6, 42, and 8(heavy chain) and 21, 11, and 12 (light chain)) are used in an anti-CD40DVD. Examples of DVD-Ig structures are known in the art and aredescribed, for example, in U.S. Pat. No. 7,612,181, which isincorporated by reference herein.

As used herein, the term “neutralizing” refers to neutralization ofbiological activity of a cytokine receptor when an antibody, or antigenbinding portion thereof, specifically binds the cytokine receptor.Preferably, a neutralizing antibody, or antigen binding portion thereof,is a neutralizing antibody whose binding to hCD40 results in inhibitionof a biological activity of hCD40. Preferably the neutralizing antibody,or antigen binding portion thereof, binds hCD40 and reduces abiologically activity of hCD40 by at least about 20%, 40%, 60%, 80%, 85%or more. Inhibition of a biological activity of hCD40 by a neutralizingantibody, or antigen binding portion thereof, can be assessed bymeasuring one or more indicators of hCD40 biological activity well knownin the art.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-hCD40 antibody that binds to an hCD40 antigen and/or theneutralizing potency of an antibody, for example, an anti-hCD40 antibodywhose binding to hCD40 inhibits the biological activity of hCD40. e.g.,binding CD40L; involvement in B cell development; involvement inlymphocyte activation; involvement in antigen presenting cells function;regulating activity of dendritic cells, macrophages and B cells;inducing production of inflammatory cytokines in macrophages anddendritic cells; up-regulating antigen presentation; up-regulating Tcell stimulation; and promoting immunoglobulin class switching in Bcells.

Exemplary assays for assessing the activity of the anti-CD40 antibodiesof the present invention include in vitro an in vivo assays as set forthherein. Specifically, the assays may be used to determine whether ananti-CD40 antibody is an agonist or an antagonist antibody.

For example, binding to human CD40 and inhibition of CD40-CD40Linteraction can be assayed using a human CD40-expressing cell line viaFACS analyses. Antagonist and agonist activities can be assessed using aCD40-expressing reporter cell line expressing human CD40 linked to NFkBmediated alkaline phosphatase (AP). When signal is received throughCD40, NFkB activation leads to secretion of AP which is measured bycolorimetric substrate. As an exemplary antagonist assay, a CD40reporter line can be cultured with either Jurkat cell line expressingCD40L (to provide physiological ligand interaction) or with solubleCD40L and the ability of anti-CD40 antibodies to block the NFkB signalcan be assessed. As an exemplary agonist assay, a human CD40 reportercell line can be treated with anti-CD40 antibodies and the NFkB signalmeasured as described above.

Alternatively, a B cell agonist assay can be utilized in which B cellsare activated with low dose anti-IgM and IL4, prior to addition of aCD40 antagonist antibody. Enhancement of B cell activation can bemeasured as upregulation of CD86, which in turn is indicative of agonistactivity. Similarly, a B cell antagonist assay can be utilized in whichprimary human B cells are cultured with CD40L-expressing human T cellline that leads to B cell activation and upregulation of CD86 expressionvia CD40/CD40L interaction. Inhibition of CD86 upregulation of primaryhuman B cells is indicative of antagonist activity.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to a an antibody or antigen-binding portion thereof. Incertain embodiments, epitope determinants include chemically activesurface groupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. In various embodiments, an epitope may be a linear orsequential epitope, i.e., a linear sequence of amino acids, of theprimary structure of the antigen, i.e., CD40. Alternatively, in otherembodiments, an epitope may be a conformational epitope having aspecific three-dimensional shape when the antigen assumes its secondarystructure. For example, the conformational epitope may comprisenon-linear, i.e., non-sequential, amino acids of the antigen.

In a particular embodiment, an epitope is a region of an antigen that isbound by an antibody or antigen-binding portion thereof. In certainembodiments, an antibody or antigen-binding portion thereof is said tospecifically bind an antigen when it preferentially recognizes itstarget antigen in a complex mixture of proteins and/or macromolecules.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in Section II C, below), antibodies isolated from a recombinant,combinatorial human antibody library (Hoogenboom H. R., (1997) TIB Tech.15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem.35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today21:371-378), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see e.g., Taylor. L. D., etal. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L.L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al(2000) Immunology Today 21:364-370) or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.One embodiment provides fully human antibodies capable of binding humanCD40 which can be generated using techniques well known in the art, suchas, but not limited to, using human Ig phage libraries such as thosedisclosed in Jermutus et al., PCT publication No. WO 2005/007699 A2.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jönsson, U., et al (1993) Ann. Biol. Clin.51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson. B., et al.(1991) Anal. Biochem. 198:268-277.

The term “k_(on)”, as used herein, is intended to refer to the on rateconstant for association of an antibody to the antigen to form theantibody/antigen complex as is known in the art.

The term “k_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex as is known in the art.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction as isknown in the art.

The term “labeled antibody” as used herein, refers to an antibody with alabel incorporated that provides for the identification of the antibody,or antigen binding portion thereof. Preferably, the label is adetectable marker, e.g., incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). Examples of labels for polypeptides include, but are notlimited to, the following: radioisotopes or radionuclides (e.g., ³H,¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm);fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic labels (e.g., horseradish peroxidase, luciferase, alkalinephosphatase); chemiluminescent markers; biotinyl groups; predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags); and magnetic agents, such as gadoliniumchelates.

The terms “crystal”, and “crystallized” as used herein, refer to anantibody, or antigen-binding portion thereof, that exists in the form ofa crystal. Crystals are one form of the solid state of matter, which isdistinct from other forms such as the amorphous solid state or theliquid crystalline state. Crystals are composed of regular, repeating,three-dimensional arrays of atoms, ions, molecules (e.g., proteins suchas antibodies), or molecular assemblies (e.g., antigen/antibodycomplexes). These three-dimensional arrays are arranged according tospecific mathematical relationships that are well-understood in thefield. The fundamental unit, or building block, that is repeated in acrystal is called the asymmetric unit. Repetition of the asymmetric unitin an arrangement that conforms to a given, well-definedcrystallographic symmetry provides the “unit cell” of the crystal.Repetition of the unit cell by regular translations in all threedimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett,Crystallization of Nucleic Acids and Proteins, a Practical Approach. 2ndea., pp. 20 1-16, Oxford University Press, New York, N.Y. (1999).”

The term “polynucleotide” as used herein refers to a polymeric form oftwo or more nucleotides, either ribonucleotides or deoxynucleotides or amodified form of either type of nucleotide. The term includes single anddouble stranded forms of DNA but preferably is double-stranded DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide (e.g., of genomic, cDNA, or synthetic origin, or somecombination thereof) that, by virtue of its origin, the “isolatedpolynucleotide”: is not associated with all or a portion of apolynucleotide with which the “isolated polynucleotide” is found innature; is operably linked to a polynucleotide that it is not linked toin nature; or does not occur in nature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences: efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence): sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Protein constructs of the presentinvention may be expressed, and purified using expression vectors andhost cells known in the art, including expression cassettes, vectors,recombinant host cells and methods for the recombinant expression andproteolytic processing of recombinant polyproteins and pre-proteins froma single open reading frame (e.g., WO 2007/014162 incorporated herein byreference).

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell, but, to the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.Preferably host cells include prokaryotic and eukaryotic cells selectedfrom any of the Kingdoms of life. Preferred eukaryotic cells includeprotist, fungal, plant and animal cells. Most preferably host cellsinclude but are not limited to the prokaryotic cell line E. coli;mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; andthe fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press. ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

“Transgenic organism”, as known in the an and as used herein, refers toan organism having cells that contain a transgene, wherein the transgeneintroduced into the organism (or an ancestor of the organism) expressesa polypeptide not naturally expressed in the organism. A “transgene” isa DNA construct, which is stably and operably integrated into the genomeof a cell from which a transgenic organism develops, directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic organism.

The term “regulate” and “modulate” are used interchangeably, and, asused herein, refers to a change or an alteration in the activity of amolecule of interest (e.g., the biological activity of hCD40).Modulation may be an increase or a decrease in the magnitude of acertain activity or function of the molecule of interest. Exemplaryactivities and functions of a molecule include, but are not limited to,binding characteristics, enzymatic activity, cell receptor activation,and signal transduction.

Correspondingly, the term “modulator.” as used herein, is a compoundcapable of changing or altering an activity or function of a molecule ofinterest (e.g., the biological activity of hCD40). For example, amodulator may cause an increase or decrease in the magnitude of acertain activity or function of a molecule compared to the magnitude ofthe activity or function observed in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of at least one activity or function of a molecule. Exemplaryinhibitors include, but are not limited to, proteins, peptides,antibodies, peptibodies, carbohydrates or small organic molecules.Peptibodies are described. e.g., in WO01/83525.

As used herein, the term “effective amount” refers to the amount of atherapy which is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof, prevent theadvancement of a disorder, cause regression of a disorder, prevent therecurrence, development, onset or progression of one or more symptomsassociated with a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

As used herein, the term “non-responder” is used to refer to a subjecthaving IBD (e.g., Crohn's disease or ulcerative colitis) who has no, orlimited or inadequate, improvement in their clinical disease statusfollowing treatment with a TNFα inhibitor (e.g., lack of reduction inCDAI score, lack of reduction in use of corticosteroids). In oneembodiment, a TNF non-responder is a subject having IBD who fails toachieve a reduction of 100 points or more in their Crohn's DiseaseActivity Index (CDAI) score following treatment with a TNFα inhibitor.In one embodiment, a non-responder is a subject having IBD who fails toachieve a reduction of 100 points or more in their Crohn's DiseaseActivity Index (CDAI) score in a specific time frame following treatmentwith a TNFα inhibitor.

I. Antibodies that Bind Human CD40 (hCD40)

One aspect of the present invention provides antagonistic humanizedantibodies, or antigen-binding portions thereof, that bind CD40,including human CD40 (hCD40). Other embodiments of the invention includemurine monoclonal antibodies, or antigen-binding portions thereof, thatbind to CD40, as well as chimeric antibodies comprising the variableregions of the anti-CD40 murine antibodies described herein. Preferably,the antibodies of the invention are antagonistic anti-CD40 (e.g.,anti-human CD40) antibodies having no significant agonist activity. 1.Humanized Anti-hCD40 Antagonist Antibodies Derived from Antibody 1 (Ab1)The invention is based, at least in part, on the identification ofhumanized anti-CD40 antibodies having antagonistic characteristics and,in certain embodiments, having no substantial agonist activity.

As described in Example 1, three antagonist anti-hCD40 murine antibodieswere identified, i.e., Ab1 (VL sequence described in SEQ ID NO: 9 and VHsequence described in SEQ ID NO: 5), Ab2 (VL sequence described in SEQID NO: 76 and VH sequence described in SEQ ID NO: 75), and Ab3 (VLsequence described in SEQ ID NO: 48 and VH sequence described in SEQ IDNO: 44).

Consensus CDR sequences were determined based on alignments of the CDRamino acid sequences of murine antagonist antibodies Ab1, Ab2, and Ab3.Consensus amino acid sequences for the VH CDR1, CDR2, and CDR3 regionsare described in SEQ ID NOs 78, 79 and 80, respectively, and consensusamino acid sequences for the VL CDR1, CDR2, and CDR3 amino acidsequences are described in SEQ ID NOs 108, 109 and 110, respectively.All sequences are also described below in Table 5 and in FIG. 4.

TABLE 5 Anti-CD40 Hybridoma CDR Amino Acid Sequence Alignments HybridomaCDR1 CDR2 CDR3 Heavy chain Ab3 GYTFTSYTMH YINPSSDYPNYNQKFKD WGYSFDY (SEQID NOS Ab1 GFTFSDYGMN YISSGRSNIYYADTVKG SWGYFDV 45-47, 6-8, Ab2GFTFSDYGMN YISSGRGNIYYADTVKG SWGYFDV 6, 42, 8 and Consensus GFTFSDYGMNYISSGR NIYYADTVKG SWGYFDV 78-80,  Y  TS T H   NPSS YPN NQKF D WGYSrespectively, in order of appearance) Light chain Ab3 RSSKSLLHS-NGNTYLYRMSTLAS MQHLEYPLT (SEQ ID NOS Ab1 KSSQSLLNSGNQKNYLT WASTRES QNDYTYPLT49-51, 10-12, Ab2 KSSQSLLNSGNQKNYLT WASTRES QNDYTYPLT 10-12 andConsensus KSSQSLLNSGNQKNYLT WASTRES QNDYTYPLT 108-110, R  K   H - GNT  YRM  LA MQHLE respectively, in order of appearance)

The consensus amino acid sequence of the heavy chain CDR1 domain is setforth as SEQ ID NO:78 (G(F/Y)TF(S/T)(D/S)Y(G/T)M(N/H)). The consensusamino acid sequence of the variable heavy chain CDR2 domain is set forthas SEQ ID NO:79 (YI(S/N)(S/P)(G/S)(R/S) (D/S/G)(N/Y)(I/P)(Y/N)Y(A/N)(D/Q)(T/K) (V/F)K(G/D)). The consensus amino acid sequence ofthe variable heavy chain CDR3 domain is set forth in SEQ ID NO:80((S/W)(W/G)(G/Y)(Y/S)FDV).

The consensus amino acid sequence of the variable light chain CDR1domain is set forth as SEQ ID NO: 108 ((K/R)SS(Q/K)SLL(N/H)S(G/-)N(Q/G)(K/N)(N/T)YL(T/Y)). The consensus amino acid sequence of the variablelight chain CDR2 domain is set for as SEQ ID NO: 109 ((W/R)(A/M) ST(R/L) (E/A)S). The consensus amino acid sequence of the variable lightchain CDR3 domain is set forth as SEQ ID NO: 110 ((Q/M)(N/Q)(D/H)(Y/L)(T/E)YPLT).

In one embodiment, the invention provides an anti-CD40 antagonistantibody, or antigen-binding portion thereof, comprising a variablelight chain comprising a CDR1 having the amino acid sequence of SEQ IDNO: 108, a CDR2 having the amino acid sequence of SEQ ID NO: 109, a CDR3having the amino acid sequence of SEQ ID NO: 110, and comprising avariable heavy chain comprising a CDR1 having the amino acid sequence ofSEQ ID NO: 78, a CDR2 having the amino acid sequence of SEQ ID NO: 79, aCDR3 having the amino acid sequence of SEQ ID NO: 80.

Following the identification of murine antibodies Ab1, Ab2, and Ab3,antibodies Ab1 and Ab3 were selected for humanization (described belowin Example 2). Tables 11 and 12 provide the amino acid sequences of CDR,VH, and VL regions of humanized Ab1 and Ab3, respectively. Specifically,nine different humanized antibodies were created based on Ab3 (seeExample 2 and Table 12 below). Four different humanized antibodies basedon Ab1 were also created, including the following:

A) huAb1VH.1/VL.1 (VH amino acid sequence set forth as SEQ ID NO: 13 andVH CDR1, CDR2, and CDR3 sequences set forth as SEQ ID NOs: 6, 7, and 8,respectively; and VL amino acid sequence set forth as SEQ ID NO: 14 andVL CDR1, CDR2, and CDR3 sequences set forth as SEQ ID NOs: 10, 11, and12, respectively);

B) huAb1VH.1A/VL.1 (VH amino acid sequence set forth as SEQ ID NO: 15and VH CDR1, CDR2, and CDR3 sequences set forth as SEQ ID NOs: 6, 7, and8, respectively; and VL amino acid sequence set forth as SEQ ID NO: 14and VL CDR1, CDR2, and CDR3 sequences set forth as SEQ ID NOs: 10, 11,and 12, respectively);

C) huAb1VH.1/VL.1A (VH amino acid sequence set forth as SEQ ID NO: 13and VH CDR1, CDR2, and CDR3 sequences set forth as SEQ ID NOs: 6, 7, and8, respectively; and VL amino acid sequence set forth as SEQ ID NO: 16and VL CDR1, CDR2, and CDR3 sequences set forth as SEQ ID NOs: 10, 11,and 12, respectively); and

D) huAb1VH.1A/VL.1A (VH amino acid sequence set forth as SEQ ID NO: 15and VH CDR1, CDR2, and CDR3 sequences set forth as SEQ ID NOs: 6, 7, and8, respectively; VL amino acid sequence set forth as SEQ ID NO: 16 andVL CDR1, CDR2, and CDR3 sequences set forth as SEQ ID NOs: 10, 11, and12, respectively).

Humanized versions of Ab1 were further modified in order to remove apotential deamidation site in the light chain CDR1. Six variant huAb1antibodies were analyzed, and four of the antibodies were identified asbeing antagonists of CD40. The six antibodies are referred to herein asAb1v1, Ab1v2, Ab1v3, Ab1v4, Ab1v5, and Ab1v6 (CDR and variable sequencesare provided in Table 13 below). Of the six humanized Ab1 variants,huAb1v1 was selected as having particularly superior anatagonistactivity. The heavy chain variable sequence of huAb1v1 is provided inSEQ ID NO: 15 with CDR1, CDR2, and CDR3 sequences described in SEQ IDNOs: 6, 7, and 8, respectively. The light chain variable sequence ofhuAb1v1 is provided in SEQ ID NO: 20 with CDR1, CDR2, and CDR3 sequencesdescribed in SEQ ID NOs: 21, 11, and 12, respectively.

The heavy chain CDR2 of antibody huAb1v1 was further mutagenizedresulting in seventeen variants (described below in Example 4). ThesehuAb1v1 heavy chain CDR2 variants are referred to herein ashuAb1v1CDR2v1 to huAb1v1CDR2v17. The sequences of the huAb1v1CDR2v1 tohuAb1v1CDR2v17 heavy chains are provided in Table 16, where VHhuAb1v1CDR2v7 was selected as the clone having particularly superiorantagonistic activity against CD40, while remaining relatively free ofagonist activity. The heavy chain variable sequence of huAb1v1CDR2v7 isprovided in SEQ ID NO: 28 with CDR1, CDR2, and CDR3 sequences describedin SEQ ID NOs: 6, 42, and 8, respectively.

Following the selection of the huAb1v1CDR2v7 VH (VH of SEQ ID NO: 28with CDR1, CDR2, and CDR3 sequences described in SEQ ID NOs: 6, 42, and8, respectively) and the huAb1v1 VL (VL of SEQ ID NO: 20 with CDR1,CDR2, and CDR3 sequences described in SEQ ID NOs: 21, 11, and 12,respectively), the variable regions were cloned into two different IgGbackgrounds resulting in two anti-CD40 antagonist antibodies, i.e.,Ab101 and Ab102. Table 6 (and Table 19) provides full length heavy chainand light chain sequences for particular embodiments of the presentinvention relating to these IgG antibodies. In Table 16, constantregions are underlined and CDR domains are in bold.

TABLE 6 Humanized Anti-CD40 Antibodies Ab101 and Ab102 and Heavy andLight Chain Sequences Thereof HC LC SEQ SEQ ID ID Ab Heavy ChainSequence NO: Light Chain Sequence NO: Ab101EVQLVESSGGLVKPGGSLRLSCAASGFTFS 39 DIVMTQSPDSLAVSLGERATINCKSSQSLL 40DYGMNWVRQAPGKGLEWIAYISSGRGNIYY NRGNQKNYLTWFQQKPGQPPKLLIYWASTRADTVKGRFTISRDNAKNSLYLQMNSLRAED ESGVPDRFSGSGSGTDFTLTISSLQAEDVATAVYYCARSWGYFDVWGQGTTVTVSSASTK VYYCQNDYTYPLTFGQGTKLEIKRTVAAPSGPSVFPLAPSSKSTSGGTAALGCLVKDYFP VFIFPPSDEQLKSGTASVVCLLNNFYPREAEPVTVSWNSGALTSGVHTFPAVLQSSGLYS KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV LSSTLTLSKADYEKHKVYACEVTHQGLSSPDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF VTKSFNRGEC LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK Ab102EVQLVESSGGLVKPGGSLRLSCAASGFTFS 41 DIVMTQSPDSLAVSLGERATINCKSSQSLL 40DYGMNWVRQAPGKGLEWIAYISSGRGNIYY NRGNQKNYLTWFQQKPGQPPKLLIYWASTRADTVKGRFTISRDNAKNSLYLQMNSLRAED ESGVPDRFSGSGSGTDFTLTISSLQAEDVATAVYYCARSWGYFDVWGQGTTVTVSSASTK VYYCQNDYTYPLTFGQGTKLEIKRTVAAPSGPSVFTLAPSSKSTSGGTAALGCLVKDYFP VFIFPPSDEQLKSGTASVVCLLNNFYPREAEPVTVSWNSGALTSGVHTFPAVLQSSGLYS KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV LSSTLTLSKADYEKHKVYACEVTHQGLSSPDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF VTKSFNRGEC LFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVLHEALHNHYTQKSLSLSPGK

Accordingly, in one embodiment, the present invention is directed to anantagonist anti-CD40 antibody comprising a light chain having the aminoacid sequence as set forth in SEQ ID NO:40 and a heavy chain having theamino acid sequence as set forth in SEQ ID NO: NO:39 (Ab101). In analternative embodiment, the present invention is directed to anantagonist anti-CD40 antibody comprising a light chain having the aminoacid sequence as set forth in SEQ ID NO:40 and a heavy chain having theamino acid sequence as set forth in SEQ ID NO: NO:41 (Ab102).

Thus, the invention includes murine, chimeric, and humanized anti-CD40antibodies having antagonist activity. In certain embodiments, thepresent invention provides an antagonistic anti-CD40 antibody, orantigen binding portion thereof, including a light chain variable regionhaving a CDR3 having an amino acid sequence as set forth in SEQ ID NO:12 and/or a heavy chain variable region having a CDR3 having an aminoacid sequence as set forth in SEQ ID NO: 8. In a particular embodiment,the antagonist anti-CD40 antibody, or antigen-binding portion thereof,includes a heavy chain CDR1 having an amino acid sequence as set forthin SEQ ID NO:6, a heavy chain CDR2 having an amino acid sequence as setforth in SEQ ID NO:42, a heavy chain CDR3 having an amino acid sequenceas set forth in SEQ ID NO:8, a light chain CDR1 having an amino acidsequence as set forth in SEQ ID NO:21, a light chain CDR2 having anamino acid sequence as set forth in SEQ ID NO: 11, and a light chainCDR3 having an amino acid sequence as set forth in SEQ ID NO: 12. In aparticular embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain variable domainhaving an amino acid sequence set forth in SEQ ID NO: 28 and a lightchain variable domain having an amino acid sequence set forth in SEQ IDNO: 20. In one embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain having an aminoacid sequence set forth in SEQ ID NO: 41; and a light chain comprisingan amino acid sequence set forth in SEQ ID NO: 40. In anotherembodiment, the antagonist anti-CD40 antibody, or antigen-bindingportion thereof, includes a heavy chain having an amino acid sequenceset forth in SEQ ID NO: 39; and a light chain comprising an amino acidsequence set forth in SEQ ID NO: 40.

Antibodies having the amino acid sequences (variable or CDR) describedin Tables 5, 6, 11, 12, 13, 14, 16, 17, 18, and 19 are included in theinvention. Accordingly, in one aspect, the present invention is directedto an antagonist anti-CD40 antibody, or antigen-binding portion thereof,having a light chain variable region comprising (a) a CDR1 having anamino acid sequence as set forth in SEQ ID NO: 10, 17, 19, or 21; (b) aCDR2 having an amino acid sequence as set forth in SEQ ID NO: 11; and(c) a CDR3 having an amino acid sequence set forth in SEQ ID NO: 12.Alternatively or in combination, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain variable regioncomprising (a) a CDR1 having an amino acid sequence as set forth in SEQID NO: 6; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 7 or 42; and (c) a CDR3 having an amino acid sequence set forthin SEQ ID NO: 8.

In a particular embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, has a light chain variable regioncomprising (a) a CDR1 having an amino acid sequence as set forth in SEQID NO: 10; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 11; and (c) a CDR3 having an amino acid sequence set forth in SEQID NO: 12; and a heavy chain variable region comprising (a) a CDR1having an amino acid sequence as set forth in SEQ ID NO: 6; (b) a CDR2having an amino acid sequence as set forth in SEQ ID NO: 7; and (c) aCDR3 having an amino acid sequence set forth in SEQ ID NO: 8.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, has a light chain variable regioncomprising (a) a CDR1 having an amino acid sequence as set forth in SEQID NO: 19; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 11; and (c) a CDR3 having an amino acid sequence set forth in SEQID NO: 12, and a heavy chain variable region comprising (a) a CDR1having an amino acid sequence as set forth in SEQ ID NO: 6; (b) a CDR2having an amino acid sequence as set forth in SEQ ID NO: 7; and (c) aCDR3 having an amino acid sequence set forth in SEQ ID NO: 8.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, has a light chain variable regioncomprising (a) a CDR1 having an amino acid sequence as set forth in SEQID NO: 17; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 11; and (c) a CDR3 having an amino acid sequence set forth in SEQID NO: 12, and a heavy chain variable region comprising (a) a CDR1having an amino acid sequence as set forth in SEQ ID NO: 6; (b) a CDR2having an amino acid sequence as set forth in SEQ ID NO: 7; and (c) aCDR3 having an amino acid sequence set forth in SEQ ID NO: 8.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, has a light chain variable regioncomprising (a) a CDR1 having an amino acid sequence as set forth in SEQID NO: 21; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 11; and (c) a CDR3 having an amino acid sequence set forth in SEQID NO: 12; and a heavy chain variable region comprising (a) a CDR1having an amino acid sequence as set forth in SEQ ID NO: 6; (b) a CDR2having an amino acid sequence as set forth in SEQ ID NO: 7; and (c) aCDR3 having an amino acid sequence set forth in SEQ ID NO: 8.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, has a light chain variable regioncomprising (a) a CDR1 having an amino acid sequence as set forth in SEQID NO: 21; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 11; and (c) a CDR3 having an amino acid sequence set forth in SEQID NO: 12; and a heavy chain variable region comprising (a) a CDR1having an amino acid sequence as set forth in SEQ ID NO: 6; (b) a CDR2having an amino acid sequence as set forth in SEQ ID NO: 42; and (c) aCDR3 having an amino acid sequence set forth in SEQ ID NO: 8.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, has a light chain variable regioncomprising (a) a CDR1 having an amino acid sequence as set forth in SEQID NO: 108; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 109; and (c) a CDR3 having an amino acid sequence set forth inSEQ ID NO: 110; and a heavy chain variable region comprising (a) a CDR1having an amino acid sequence as set forth in SEQ ID NO: 78; (b) a CDR2having an amino acid sequence as set forth in SEQ ID NO: 79; and (c) aCDR3 having an amino acid sequence set forth in SEQ ID NO: 80.

In yet another aspect of the present invention, an antagonist anti-CD40antibody, or antigen binding portion thereof, comprises a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNOs: 5, 13, 15, or 22-38; and/or a light chain variable region includingan amino acid sequence as set forth in SEQ ID NOs: 9, 14, 16, 18, 20 or43.

In a particular embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain variable regionincluding an amino acid sequence as set forth in SEQ ID NO: 5 and alight chain variable region including an amino acid sequence as setforth in SEQ ID NO: 9.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain variable regionincluding an amino acid sequence as set forth in SEQ ID NO: 13 and alight chain variable region including an amino acid sequence as setforth in SEQ ID NO: 14, 16 or 18.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain variable regionincluding an amino acid sequence as set forth in SEQ ID NO: 15 and alight chain variable region including an amino acid sequence as setforth in SEQ ID NO:14, 16, 18, 20 or 43.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain variable regionincluding an amino acid sequence as set forth in SEQ ID NO: 28 and alight chain variable region including an amino acid sequence as setforth in SEQ ID NO: 20.

The antibodies described herein, particularly antibody Ab102, haveantagonistic activity without substantial agonist activity for CD40.Thus, included in the invention are antibodies that bind to the epitoperecognized by antibodies Ab102 and Ab101. In a particular embodiment,the invention includes an isolated antibody, or antigen binding portionthereof, wherein said antibody, or antigen binding fragment thereof,binds human CD40 such that CD40 with said antibody, or antigen bindingfragment thereof, bound to an epitope defined by the topographic regionsCys62-Phe67, Gln79-Cys83, Arg90-Thr99, and Thr24-Cys37 of SEQ ID NO:1 isinhibited from binding to the CD40 ligand (CD40L). In another aspect,the invention pertains to an antibody, or antigen binding fragmentthereof, capable of binding human CD40 that binds to an epitope in humanCD40 comprising three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four,twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine,thirty, thirty-one, thirty-two, thirty-three, thirty-four, or all of theamino acid residues Cys62-Phe67 (Cys62, Gly63, Glu64, Ser65, Glu66,Phe67), Gln79-Cys83 (Gln79, His 80, Lys81, Tyr82, Cys83), Arg90-Thr99(Arg90, Val91, Gln92, Gln93, Lys94, Gly95, Thr96, Ser97, Glu98 andThr99), and Thr24-Cys37 (Thr24, Ala25, Cys26, Arg27, Glu28, Lys29,Gln30, Tyr31, Leu32, Ile33, Asn34, Ser35, Gln36. Cys37) of SEQ ID NO: 1.

In another embodiment, the present invention provides a heavy chain CDR2region having antagonist activity against CD40. In particular, residue55 (residue X) of the VH CDR2 amino acid sequence YISSGRXNIYYADTVKG (SEQID NO: 112) has been identified as playing a role in increasingantagonist activity of the antibody relative to a parent CDR2 sequencehaving residue S55. Residues Thr, Asp, Val, Leu, Ile, and Met atposition 55 of the HC CDR2 result in lower levels of antagonisticactivity relative to other amino acids at position 55. In oneembodiment, the invention provides an antagonist anti-CD40 antibody, orantigen binding portion thereof, comprising a heavy chain variableregion comprising a CDR2 having the amino acid sequence of SEQ ID NO:111 and a light chain variable region comprising a CDR2 having the aminoacid sequence of SEQ ID NO: 11. CDR1 and CDR3 domain amino acidsequences that may be combined with SEQ ID NO: 111 in variable heavy andlight antibody chains are described throughout, including, for example,Tables 13 and 18.

In a further embodiment, the present invention provides a light chainCDR1 region having a residue identified as an antagonist/agonist switch.As described below in Example 3, modification of the “NS” motif of VLCDR1 region KSSQSLLNSGNQKNYLT (SEQ ID NO: 10) at residue “S” may resultin an antagonist antibody switching to an agonist antibody. Thus, in oneembodiment, the invention includes an antagonist anti-CD40 antibodyhaving a CDR1 VL region comprising SEQ ID NO: 113 (KSSQSLLNXGNQKNYLT;wherein X is not amino acid residue Pro).

The term “competing antibodies” herein refers to any number ofantibodies targeting the same molecular or stably but non-covalentlylinked supermolecular entity, preferably the same molecule, i.e., CD40,wherein at least one is capable of specifically reducing the measurablebinding of another, preferably by sterically hampering the other'saccess to its target epitope (described above) or by inducing and/orstabilizing a conformation in the target entity that reduces thetarget's affinity for the other antibody, more preferably by directlyblocking access to the other's target epitope by binding to an epitopein sufficiently close vicinity of the former, overlapping with theformer or identical to the former, most preferably overlapping oridentical, in particular identical. Two epitopes are herein said to be“overlapping” if they share part of their chemical structures,preferably their amino acid sequences, and to be “identical”, if theirchemical structures, preferably their amino acid sequences, areidentical.

In particular embodiments, the competing antibody, or antigen-bindingportion thereof, is an antibody, or antigen-binding portion thereof,that competes with any of the antibodies presented herein. In oneembodiment, the invention provides a competing antibody which cancompete with antibodies described herein (e.g., Ab101 or Ab102) andbinds to a topographical epitope of human CD40 including residuesCys62-Phe67, Gln79-Cys83. Arg90-Thr99, and Thr24-Cys37.

In particular embodiments, the agonist antibody comprises a heavy chainvariable region including (a) a CDR1 having an amino acid sequence setforth in SEQ ID NO: 6; (b) a CDR2 having an amino acid sequence as setforth in SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequence setforth in SEQ ID NO: 8; and a light chain variable region including (a) aCDR1 having an amino acid sequence as set forth in SEQ ID NO: 74; (b) aCDR2 having an amino acid sequence as set forth in SEQ ID NO: 11; and(c) a CDR3 having an amino acid sequence set forth in SEQ ID NO: 12.

In other particular embodiments, the agonist antibody comprises a heavychain variable region including (a) a CDR1 having an amino acid sequenceset forth in SEQ ID NO: 6; (b) a CDR2 having an amino acid sequence asset forth in SEQ ID NO: 7; and (c) a CDR3 having an amino acid sequenceset forth in SEQ ID NO: 8; and a light chain variable region including(a) a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 17;(b) a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 11;and (c) a CDR3 having an amino acid sequence set forth in SEQ ID NO: 12.

In further embodiments, an agonist anti-CD40 antibody, or antigenbinding portion thereof, comprises a heavy chain variable regionincluding an amino acid sequence as set forth in SEQ ID NOs: 15 or 13;and/or a light chain variable region including an amino acid sequence asset forth in SEQ ID NOs: 77 or 43.

2. Humanized Anti-CD40 Antibodies Derived from Antibody 3 (Ab3)

Amino acid sequences for humanized versions of heavy and light chains ofmurine Ab3 are provided in Table 11 below. Thus, the invention furtherfeatures antibodies comprising the variable and/or CDR sequences fromantibody 3 (Ab3).

In one aspect, the present invention provides a humanized antibody, orantigen binding portion thereof, including a light chain variable regionincluding (a) a CDR1 having an amino acid sequence as set forth in SEQID NO:49; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 50; and (c) a CDR3 having an amino acid sequence set forth in SEQID NO: 51; and a heavy chain variable region including (a) a CDR1 havingan amino acid sequence as set forth in SEQ ID NO: 45; (b) a CDR2 havingan amino acid sequence as set forth in SEQ ID NO: 46; and (c) a CDR3having an amino acid sequence set forth in SEQ ID NO: 47.

Accordingly, in one aspect, the present invention is directed to anantagonist anti-CD40 antibody, or antigen-binding portion thereof, has alight chain variable region including (a) a CDR1 having an amino acidsequence as set forth in SEQ ID NO:49; (b) a CDR2 having an amino acidsequence as set forth in SEQ ID NO: 50; and (c) a CDR3 having an aminoacid sequence set forth in SEQ ID NO: 51. Alternatively or incombination, the antagonist anti-CD40 antibody, or antigen-bindingportion thereof, includes a heavy chain variable region including (a) aCDR1 having an amino acid sequence as set forth in SEQ ID NO: 45; (b) aCDR2 having an amino acid sequence as set forth in SEQ ID NO: 46; and(c) a CDR3 having an amino acid sequence set forth in SEQ ID NO: 47.

In yet another aspect of the present invention, an antagonist anti-CD40antibody, or antigen binding portion thereof, comprises a heavy chainvariable region including an amino acid sequence as set forth in SEQ IDNOs: 44, 52, 54 or 55; and/or a light chain variable region including anamino acid sequence as set forth in SEQ ID NOs: 48, 53, 56 or 57.

In a particular embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain variable regionincluding an amino acid sequence as set forth in SEQ ID NO: 44 and alight chain variable region including an amino acid sequence as setforth in SEQ ID NO: 48.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain variable regionincluding an amino acid sequence as set forth in SEQ ID NO: 52 and alight chain variable region including an amino acid sequence as setforth in SEQ ID NO:53, 56 or 57.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain variable regionincluding an amino acid sequence as set forth in SEQ ID NO: 54 and alight chain variable region including an amino acid sequence as setforth in SEQ ID NO:53, 56 or 57.

In another embodiment, the antagonist anti-CD40 antibody, orantigen-binding portion thereof, includes a heavy chain variable regionincluding an amino acid sequence as set forth in SEQ ID NO: 55 and alight chain variable region including an amino acid sequence as setforth in SEQ ID NO:53, 56 or 57.

3. Anti CD40 Chimeric Antibodies

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and ahuman immunoglobulin constant region. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and4,816,397, which are incorporated herein by reference in theirentireties. In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454, each of which are incorporated herein byreference in their entireties) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.

In another aspect, the present invention is directed to an antagonistanti-CD40 antibody, or antigen-binding portion thereof, having a lightchain variable region including (a) a CDR1 having an amino acid sequenceas set forth in SEQ ID NO: 10; (b) a CDR2 having an amino acid sequenceas set forth in SEQ ID NO: 11; and (c) a CDR3 having an amino acidsequence set forth in SEQ ID NO: 12; and a heavy chain variable regionincluding (a) a CDR1 having an amino acid sequence as set forth in SEQID NO: 6; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 42; and (c) a CDR3 having an amino acid sequence set forth in SEQID NO: 8. In a particular embodiment, the antagonist anti-CD40 antibody,or antigen binding portion thereof, has a light chain variable regionincluding the amino acid sequence set forth in SEQ ID NO:76 and a heavychain variable region including the amino acid sequence set forth in SEQID NO:75.

In another aspect, the present invention is directed to an antagonistanti-CD40 antibody, or antigen-binding portion thereof, having a lightchain variable region including (a) a CDR1 having an amino acid sequenceas set forth in SEQ ID NO:49; (b) a CDR2 having an amino acid sequenceas set forth in SEQ ID NO: 50; and (c) a CDR3 having an amino acidsequence set forth in SEQ ID NO: 51; and a heavy chain variable regionincluding (a) a CDR1 having an amino acid sequence as set forth in SEQID NO: 45; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 46; and (c) a CDR3 having an amino acid sequence set forth in SEQID NO: 47. In a particular embodiment, the antagonist anti-CD40antibody, or antigen binding portion thereof, has a light chain variableregion including the amino acid sequence set forth in SEQ ID NO:48 and aheavy chain variable region including the amino acid sequence set forthin SEQ ID NO:44.

In another aspect, the present invention is directed to an antagonistanti-CD40 antibody, or antigen-binding portion thereof, having a lightchain variable region including (a) a CDR1 having an amino acid sequenceas set forth in SEQ ID NO:10; (b) a CDR2 having an amino acid sequenceas set forth in SEQ ID NO: 11; and (c) a CDR3 having an amino acidsequence set forth in SEQ ID NO: 12; and a heavy chain variable regionincluding (a) a CDR1 having an amino acid sequence as set forth in SEQID NO: 6; (b) a CDR2 having an amino acid sequence as set forth in SEQID NO: 7; and (c) a CDR3 having an amino acid sequence set forth in SEQID NO: 8. In a particular embodiment, the antagonist anti-CD40 antibody,or antigen binding portion thereof, has a light chain variable regionincluding the amino acid sequence set forth in SEQ ID NO:9 and a heavychain variable region including the amino acid sequence set forth in SEQID NO:5.

The foregoing isolated anti-CD40 antibody CDR sequences establish anovel family of CD40 antibodies, or antigen binding portions thereof,isolated in accordance with this invention, and including antibodiesthat include the CDR sequences listed in Tables 5, 11-13, and 15-18. Togenerate and to select CDRs of the invention having preferred CD40binding and/or neutralizing activity with respect to hCD40, standardmethods known in the art for generating antibodies of the presentinvention and assessing the CD40 binding and/or neutralizingcharacteristics of those antibodies may be used, including but notlimited to those specifically described herein.

4. Characterizing Antibodies of the Invention

Anti-CD40 antibodies of the present invention are antagonist antibodiesand may exhibit a high capacity to reduce or to neutralize CD40activity, e.g., as assessed by any one of several in vitro and in vivoassays known in the art and as described herein. In certain embodiments,the anti-CD40 antibodies of the invention are antagonistic and aresubstantially free of agonist activity. Antagonist and agonist activitymay be determined by assays known in the art, including those describedherein. For example, binding to human CD40 and inhibition of CD40-CD40Linteraction can be assayed using a human CD40-expressing cell line viaFACS analyses.

In one embodiment, CD40 antagonist or agonist activity of an anti-CD40antibody is determined using a reporter cell line. For example,antagonist and agonist activities can be assessed using aCD40-expressing reporter cell line expressing human CD40 linked to NFkBmediated alkaline phosphatase (AP). When signal is received throughCD40, NFkB activation leads to secretion of AP which is measured bycolorimetric substrate. As an exemplary antagonist assay, a CD40reporter line can be cultured with either Jurkat cell line expressingCD40L (to provide physiological ligand interaction) or with solubleCD40L, where the ability of the anti-CD40 antibody to block the NFkBsignal (little to no presence of AP as determined by standard methods)can be assessed. As an exemplary agonist assay, a human CD40 reportercell line can be treated with anti-CD40 antibodies and the NFkB signalmeasured (seen as AP presence above a negative control) as describedabove. An example of a CD40 reporter cell line is HEK-Blue™ CD40L Cells(InvivoGen), which serve to measure the bioactivity of CD40L throughsecretion of embryonic alkaline phosphatase (SEAP) upon NF-κB activationfollowing CD40 stimulation. CD40L-CD40 interaction can be monitored byassessing the levels of SEAP using QUANTI-Blue (InvivoGen).

In one embodiment, an anti-CD40 antagonist antibody, or antigen-bindingfragment thereof, of the invention has an IC50 of 0.4 nM or less, asdetermined by an antagonist soluble CD40L reporter assay. In oneembodiment, an anti-CD40 antagonist antibody, or antigen-bindingfragment thereof, of the invention has an IC50 of 51 nM or less, asdetermined by an antagonist CD40 reporter assay in a Jurkat cell line.In one embodiment, an anti-CD40 antagonist antibody, or antigen-bindingfragment thereof, of the invention has an IC50 of 3.4 nM or less, asdetermined by an antagonist CD40 reporter assay in a Jurkat cell line.In one embodiment, an anti-CD40 antagonist antibody, or antigen-bindingfragment thereof, of the invention has an IC50 of 0.9 nM or less, asdetermined by an antagonist CD40 reporter assay in a Jurkat cell line.

In one embodiment, CD40 antagonist or agonist activity of an anti-CD40antibody is determined using a B cell agonist assay. For example, a Bcell agonist assay can be utilized in which B cells are activated withlow dose anti-IgM and IL4, prior to addition of a CD40 antagonistantibody. Enhancement of B cell activation can be measured asupregulation of CD86, which in turn is indicative of agonist activity.Similarly, a B cell antagonist assay can be utilized in which primaryhuman B cells are cultured with CD40L-expressing human T cell line thatleads to B cell activation and upregulation of CD86 expression viaCD40/CD40L interaction. Inhibition of CD86 upregulation of primary humanB cells is indicative of antagonist activity.

In one embodiment. CD40 antagonist or agonist activity of an anti-CD40antibody is determined using an antibody-dependent cell-mediatedcytotoxicity (ADCC) mediated assay. Antagonist and agonist activitiescan be assessed by the ability of the antibody to mediate ADCC. Anantagonistic CD40 antibody will be an effective mediator of ADCC,whereas an agonist antibody will not have ADCC activity.Antibody-Dependent Cellular Cytotoxicity (ADCC) refers to a type ofcytotoxicity induced by activation of macrophages, NK cells, neutrophilcells, etc., which recognize target cells by binding to the constantregion of the antibody via Fc receptors expressed on their surface.Complement-Dependent Cytotoxicity (CDC) refers to a type of cytotoxicityinduced by activation of a complement system which occurs throughbinding of an antibody to an antigen. A reduction in ADCC and CDCactivities means reduction in those activities as compared with, forexample, a control anti-CD40 antagonist antibody such as the monoclonalantibody produced by hybridoma 4D11 (Accession No. FERM BP-7758).EP1707627B1, incorporated by reference in its entirety herein, describesassays to determine ADCC and CDC activity.

Additionally, the biological activity of dendritic cells (DCs)stimulated with immobilized anti-CD40 Ab can be used to assay agonistactivity. DCs are believed to be the most potent antigen presentingcells that are capable of picking up Ags in nonlymphoid tissues andcarrying them to secondary lymphoid organs to prime T cells in responseto maturation stimuli such as danger and help signals. By contrast, thepresentation of Ags by DCs without activation results in the eliminationof effector T cells that have a cognate TCR or induction of regulatory Tcells in secondary lymphoid tissues. Thus, the presence or absence ofmaturation signals for immature DCs in peripheral tissues acts as aswitch to induce either an adaptive immune response or tolerance. Themajor CD4+ T cell help signal for DC maturation is provided by theinteraction between CD40 expressed on DCs and CD40 ligand (L) onactivated CD4+ T cells. Thus, CD40 stimulation induces the migration ofDCs into secondary lymphoid tissues by up-regulating the expression ofCCR7. Watanabe et al. 2003 J Immunol; 171:5828-5836 describes assayswhich may be used to determine whether anti-CD40 antibodies can activateDCs in order to determine agonistic and antagonistic activity. Suchexemplary assays include determination of the expression of MHC classI/II Ag and costimulatory molecules on BM-DCs stimulated withimmobilized anti-CD40 Ab, in vivo migration activity of BM-DCsstimulated with immobilized anti-CD40 Abs, in vitro migration activityand CCR7 expression of BM-DCs stimulated with immobilized anti-CD40 Ab.

In one embodiment, agonist activity of an anti-CD40 antibody, or antigenbinding portion thereof, is determined using an in vitro monocyteactivation assay, such as the assay described below in Example 7. An invitro monoctyte activation assay includes exposing monocytes to ananti-CD40 antibody, or antigen-binding portion thereof, where if theantibody, or antigen-binding portion thereof, is an activator of CD40(agonist) then there is a resulting increase in TNF production. Using anin vitro monocyte activation assay, an antagonist anti-CD40 antibody, orantigen binding portion thereof, that is substantially free of agonistactivity would result in the absence or minimal production of TNF, asdescribed in Example 7.

In one embodiment, an antagonist anti-CD40 antibody, or antigen bindingportion thereof, that is substantially free of agonist activity hasactivity that is within one standard deviation of a negative control inan in vitro CD40 agonist assay. e.g., agonist monocyte assay describedin Example 7.

Agonist assays are further described in U.S. Pat. No. 5,786,456,US2011/0243932 and EP1707627B1, each of which are incorporated byreference in their entireties herein. Antagonist assays for testingantibody function are further described in U.S. Pat. No. 7,361,345,US2011/0243932 and EP1707627B1, each of which is incorporated byreference herein.

In preferred embodiments, the isolated antibody, or antigen-bindingportion thereof, binds human CD40, wherein the antibody, orantigen-binding portion thereof, dissociates from human CD40 with ak_(off) rate constant of about 0.1 s⁻¹ or less, as determined by surfaceplasmon resonance, or which inhibits human CD40 activity with an IC₅₀ ofabout 1×10⁻⁶M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from human CD40 with ak_(off) rate constant of about 1×10⁻²s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit human CD40 activity with anIC₅₀ of about 1×10⁻⁷M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from human CD40 with ak_(off) rate constant of about 1×10⁻³s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit human CD40 with an IC₅₀ ofabout 1×10⁻⁸M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from human CD40 with ak_(off) rate constant of about 1×10⁻⁴s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit CD40 activity with an IC₅₀ ofabout 1×10⁻⁹M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from human CD40 with ak_(of) rate constant of about 1×10⁻⁵s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit CD40 with an IC₅₀ of about1×10⁻¹⁰M or less. Alternatively, the antibody, or an antigen-bindingportion thereof, may dissociate from human CD40 with a k_(off) rateconstant of about 1×10⁻⁵s⁻¹ or less, as determined by surface plasmonresonance, or may inhibit CD40 activity with an IC₅₀ of about 1×10⁻¹¹Mor less.

The antibodies were humanized as described in the Examples below.Framework back-mutations were introduced into the CDR-grafted antibodysequences by de novo synthesis of the variable domain or by mutagenicoligonucleotide primers and polymerase chain reaction, or by bothallowing different combinations of back mutations and other mutationsfor each of the CDR-grafts. The humanized variable regions of the murinemonoclonal CD40 antibodies were cloned into IgG expression vectors forfunctional characterization.

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantregion. Preferably, the heavy chain constant region is an IgG1 heavychain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

5. Generation of Anti-CD40 Humanized Antibodies

As described above, humanized antibodies are antibody molecules fromnon-human species antibody that binds the desired antigen having one ormore complementarity determining regions (CDRs) from the non-humanspecies and framework regions from a human immunoglobulin molecule.Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/.about.pedro/research_tools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH-05/kuby05.htm;www.library.thinkquest.org/2429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html;www.antibodyresource.com/;mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/;pathbox.wustl.edul.about.hcenter/index.-html;www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html-;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;www.biodesign.com/table.asp; www.icnet.uklaxp/facs/davies/links.html;ww.biotech.ufl.edu/.about.fccliprotocol.html;www.isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;baserv.uci.kun.nL.about.jraats/linksl.html;www.recab.uni-hd.de/immuno.bme.nwu.edu/; www.mrc-cpe.cam.ac.ukimt-doc/public/INTRO.html; www.ibt.unam.mx/vir/V_mice.html;imgt.cnusc.fr:8104/; www.biochem.ucl.ac.uk/.about.martin/absiindex.html;antibodv.bath.ac.uki; abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.ch/.about.honegger/AHOseminar/Slide01.html;www.cryst.bbk.ac.ukl.about.ubcg07s/;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk.about.mrc7/humanisation/TAHHP.html;ww,w.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk.about.fmolina/Webpages/Pept/spottech.html;www.jerini.de/fr roducts.htm; www.patents.ibm.comiibm.html.Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way. FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska, et al., PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400,U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, eachentirely incorporated herein by reference, included references citedtherein.

Examples of anti-CD40 humanized antibodies are provided in Sections 1and 2 above and in the Examples below.

II. Production of Antibodies and Antibody-Producing Cell Lines

Antibodies of the present invention may be made by any of a number oftechniques known in the art.

1. Anti-CD40 Monoclonal Antibodies Using Hybridoma Technology

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al; Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981) (said references incorporated byreference in their entireties). The term “monoclonal antibody” as usedherein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In oneembodiment, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention (See Example 1). Briefly, mice canbe immunized with a CD40 antigen. In a preferred embodiment, the CD40antigen is administered with an adjuvant to stimulate the immuneresponse. Such adjuvants include complete or incomplete Freund'sadjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulatingcomplexes). Such adjuvants may protect the polypeptide from rapiddispersal by sequestering it in a local deposit, or they may containsubstances that stimulate the host to secrete factors that arechemotactic for macrophages and other components of the immune system.Preferably, if a polypeptide is being administered, the immunizationschedule will involve two or more administrations of the polypeptide,spread out over several weeks.

After immunization of an animal with a CD40 antigen, antibodies and/orantibody-producing cells may be obtained from the animal. An anti-CD40antibody-containing serum is obtained from the animal by bleeding orsacrificing the animal. The serum may be used as it is obtained from theanimal, an immunoglobulin fraction may be obtained from the serum, orthe anti-CD40 antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected. e.g., antibodies specific for theantigen CD40 are detected in the mouse serum, the mouse spleen isharvested and splenocytes isolated. The splenocytes are then fused bywell-known techniques to any suitable myeloma cells, for example cellsfrom cell line SP20 available from the ATCC. Hybridomas are selected andcloned by limited dilution. The hybridoma clones are then assayed bymethods known in the art for cells that secrete antibodies capable ofbinding CD40. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow and Lane, supra. Ina preferred embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using CD40, or a portion thereof,or a cell expressing CD40. In a preferred embodiment, the initialscreening is performed using an enzyme-linked immunoassay (ELISA) or aradioimmunoassay (RIA), preferably an ELISA. An example of ELISAscreening is provided in WO 00/37504, herein incorporated by reference.

Anti-CD40 antibody-producing hybridomas are selected, cloned and furtherscreened for desirable characteristics, including robust hybridomagrowth, high antibody production and desirable antibody characteristics,as discussed further below. Hybridomas may be cultured and expanded invivo in syngeneic animals, in animals that lack an immune system, e.g.,nude mice, or in cell culture in vitro. Methods of selecting, cloningand expanding hybridomas are well known to those of ordinary skill inthe art.

In a preferred embodiment, the hybridomas are mouse hybridomas, asdescribed above. In another preferred embodiment, the hybridomas areproduced in a non-human, non-mouse species such as rats, sheep, pigs,goats, cattle or horses. In another embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an anti-CD40 antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example. Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

2. Anti-CD40 Monoclonal Antibodies Using SLAM

In another aspect of the invention, recombinant antibodies are generatedfrom single, isolated lymphocytes using a procedure referred to in theart as the selected lymphocyte antibody method (SLAM), as described inU.S. Pat. No. 5,627,052, PCT Publication WO 92/02551 and Babcock, J. S.et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method,single cells secreting antibodies of interest, e.g., lymphocytes derivedfrom any one of the immunized animals described in Section 1, arescreened using an antigen-specific hemolytic plaque assay, wherein theantigen CD40, a subunit of CD40, or a fragment thereof, is coupled tosheep red blood cells using a linker, such as biotin, and used toidentify single cells that secrete antibodies with specificity for CD40.Following identification of antibody-secreting cells of interest, heavy-and light-chain variable region cDNAs are rescued from the cells byreverse transcriptase-PCR and these variable regions can then beexpressed, in the context of appropriate immunoglobulin constant regions(e.g., human constant regions), in mammalian host cells, such as COS orCHO cells. The host cells transfected with the amplified immunoglobulinsequences, derived from in vivo selected lymphocytes, can then undergofurther analysis and selection in vitro, for example by panning thetransfected cells to isolate cells expressing antibodies to CD40. Theamplified immunoglobulin sequences further can be manipulated in vitro,such as by in vitro affinity maturation methods such as those describedin PCT Publication WO 97/29131 and PCT Publication WO 00/56772.

3. Anti-CD40 Monoclonal Antibodies Using Transgenic Animals

In another embodiment of the instant invention, antibodies are producedby immunizing a non-human animal comprising some, or all, of the humanimmnunoglobulin locus with a CD40 antigen. In a preferred embodiment,the non-human animal is a XENOMOUSE transgenic mouse, an engineeredmouse strain that comprises large fragments of the human immunoglobulinloci and is deficient in mouse antibody production. See, e.g., Green etal. Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771,5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and6,130,364. See also WO 91/10741, published Jul. 25, 1991, WO 94/02602,published Feb. 3, 1994, WO 96/34096 and WO 96/33735, both published Oct.31, 1996, WO 98/16654, published Apr. 23, 1998, WO 98/24893, publishedJun. 11, 1998, WO 98/50433, published Nov. 12, 1998, WO 99/45031,published Sep. 10, 1999, WO 99/53049, published Oct. 21, 1999, WO 0009560, published Feb. 24, 2000 and WO 00/037504, published Jun. 29,2000. The XENOMOUSE transgenic mouse produces an adult-like humanrepertoire of fully human antibodies, and generates antigen-specifichuman Mabs. The XENOMOUSE transgenic mouse contains approximately 80% ofthe human antibody repertoire through introduction of megabase sized,germline configuration YAC fragments of the human heavy chain loci and xlight chain loci. See Mendez et al., Nature Genetics 15:146-156 (1997),Green and Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures ofwhich are hereby incorporated by reference.

4. Anti-CD40 Monoclonal Antibodies Using Recombinant Antibody Libraries

In vitro methods also can be used to make the antibodies of theinvention, wherein an antibody library is screened to identify anantibody having the desired binding specificity.

Methods for such screening of recombinant antibody libraries are wellknown in the art and include methods described in, for example, Ladneret al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al.PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum Antibody Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982, US patent applicationpublication 20030186374, and PCT Publication No. WO 97/29131, thecontents of each of which are incorporated herein by reference.

The recombinant antibody library may be from a subject immunized withCD40, or a portion of CD40, such as the extracellular domain.Alternatively, the recombinant antibody library may be from a naïvesubject, i.e., one who has not been immunized with CD40, such as a humanantibody library from a human subject who has not been immunized withhuman CD40. Antibodies of the invention are selected by screening therecombinant antibody library with the peptide comprising human CD40 tothereby select those antibodies that recognize CD40. Methods forconducting such screening and selection are well known in the art, suchas described in the references in the preceding paragraph. To selectantibodies of the invention having particular binding affinities forhCD40, such as those that dissociate from human CD40 with a particulark_(off) rate constant, the art-known method of surface plasmon resonancecan be used to select antibodies having the desired k_(off) rateconstant. To select antibodies of the invention having a particularneutralizing activity for hCD40, such as those with a particular anIC₅₀, standard methods known in the art for assessing the inhibition ofhCD40 activity may be used.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human CD40. Preferably, theantibody is a neutralizing antibody. In various embodiments, theantibody is a recombinant antibody or a monoclonal antibody.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619: WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484, 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification of dualspecificity antibodies of the invention. One type of alternativeexpression system is one in which the recombinant antibody library isexpressed as RNA-protein fusions, as described in PCT Publication No. WO98/31700 by Szostak and Roberts, and in Roberts. R. W. and Szostak, J.W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, acovalent fusion is created between an mRNA and the peptide or proteinthat it encodes by in vitro translation of synthetic mRNAs that carrypuromycin, a peptidyl acceptor antibiotic, at their 3′ end. Thus, aspecific mRNA can be enriched from a complex mixture of mRNAs (e.g., acombinatorial library) based on the properties of the encoded peptide orprotein, e.g., antibody, or portion thereof, such as binding of theantibody, or portion thereof, to the dual specificity antigen. Nucleicacid sequences encoding antibodies, or portions thereof, recovered fromscreening of such libraries can be expressed by recombinant means asdescribed above (e.g., in mammalian host cells) and, moreover, can besubjected to further affinity maturation by either additional rounds ofscreening of mRNA-peptide fusions in which mutations have beenintroduced into the originally selected sequence(s), or by other methodsfor affinity maturation in vitro of recombinant antibodies, as describedabove.

In another approach the antibodies of the present invention can also begenerated using yeast display methods known in the art. In yeast displaymethods, genetic methods are used to tether antibody domains to theyeast cell wall and display them on the surface of yeast. In particular,such yeast can be utilized to display antigen-binding domains expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Examples of yeast display methods that can be used to make theantibodies of the present invention include those disclosed in Wittrupet al. (U.S. Pat. No. 6,699,658) incorporated herein by reference.

5. Production of Recombinant CD40 Antibodies

Antibodies of the present invention may be produced by any of a numberof techniques known in the art. For example, expression from host cells,wherein expression vector(s) encoding the heavy and light chains is(are) transfected into a host cell by standard techniques. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell. e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is possible to express the antibodies of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells is preferable, and most preferable in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr− CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl.Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g.,as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621). NS0 myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody of this invention. Recombinant DNAtechnology may also be used to remove some, or all, of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention and theother heavy and light chain are specific for an antigen other than theantigens of interest by crosslinking an antibody of the invention to asecond antibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr− CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

Another embodiment of the invention provides a glycosylated antibody, oran antigen-binding portion thereof, wherein the antibody orantigen-binding portion thereof comprises one or more carbohydrateresidues. Nascent in vivo protein production may undergo furtherprocessing, known as post-translational modification. In particular,sugar (glycosyl) residues may be added enzymatically, a process known asglycosylation. The resulting proteins bearing covalently linkedoligosaccharide side chains are known as glycosylated proteins orglycoproteins. Antibodies are glycoproteins with one or morecarbohydrate residues in the Fc domain, as well as the variable domain.Carbohydrate residues in the Fc domain have important effect on theeffector function of the Fc domain, with minimal effect on antigenbinding or half-life of the antibody (R. Jefferis, Biolechnol. Prog. 21(2005), pp. 11-16). In contrast, glycosylation of the variable domainmay have an effect on the antigen binding activity of the antibody.Glycosylation in the variable domain may have a negative effect onantibody binding affinity, likely due to steric hindrance (Co, M. S., etal., Mol. Immunol. (1993) 30:1361-1367), or result in increased affinityfor the antigen (Wallick, S. C., et al., Exp. Med. (1988) 168:1099-1109;Wright, A., et al., EMBO J. (1991) 10:2717-2723).

One aspect of the present invention is directed to generatingglycosylation site mutants in which the O- or N-linked glycosylationsite of the antibody, or an antigen-binding portion thereof, has beenmutated. One skilled in the art can generate such mutants using standardwell-known technologies. Glycosylation site mutants that retain thebiological activity, but have increased or decreased binding activity,are another object of the present invention.

In still another embodiment, the glycosylation of the antibody orantigen-binding portion of the invention is modified. For example, anaglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos.5,714,350 and 6,350,861, each of which is incorporated herein byreference in its entirety.

Additionally or alternatively, a modified antibody of the invention canbe made that has an altered type of glycosylation, such as ahypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields. R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 03/035835: WO 99/54342 80, each of whichis incorporated herein by reference in its entirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. Preferably the glycosylatedantibody, or an antigen-binding portion thereof, comprises glycosylresidues such that the glycosylation pattern is human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vive after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S. patent PublicationNos. 20040018590 and 20020137134 and PCT publication WO2005100584 A2).

In addition to the antibodies, or antigen binding portions thereof, thepresent invention is also directed to an anti-idiotypic (anti-Id)antibody specific for such antibodies, or antigen binding portionsthereof, of the invention. An anti-Id antibody is an antibody, whichrecognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal with the antibody, or antigen binding portionthereof, or a CDR containing region thereof. The immunized animal willrecognize, and respond to the idiotypic determinants of the immunizingantibody and produce an anti-Id antibody. The anti-Id antibody may alsobe used as an “immunogen” to induce an immune response in yet anotheranimal, producing a so-called anti-anti-Id antibody.

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.Preferably, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

III. Uses of Antagonistic Anti-CD40 Antibodies

Given their ability to bind to human CD40, the anti-human CD40antibodies, or portions thereof, of the invention can be used to detecthuman CD40 (e.g., in a biological sample, such as serum or plasma),using a conventional immunoassay, such as an enzyme linked immunosorbentassays (ELISA), an radioimmunoassay (RIA) or tissueimmunohistochemistry. The invention provides a method for detectinghuman CD40 in a biological sample comprising contacting a biologicalsample with an antibody, or antibody portion, of the invention anddetecting either the antibody (or antibody portion) bound to human CD40or unbound antibody (or antibody portion), to thereby detect human CD40in the biological sample. The antibody is directly or indirectly labeledwith a detectable substance to facilitate detection of the bound orunbound antibody. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescent materialsand radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin-biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ³H, ¹⁴C,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

Alternative to labeling the antibody, human CD40 can be assayed inbiological fluids by a competition immunoassay utilizing rhCD40standards labeled with a detectable substance and an unlabeledanti-human CD40 antibody. In this assay, the biological sample, thelabeled rhCD40 standards and the anti-human CD40 antibody are combinedand the amount of labeled rhCD40 standard bound to the unlabeledantibody is determined. The amount of human CD40 in the biologicalsample is inversely proportional to the amount of labeled rhCD40standard bound to the anti-CD40 antibody. Similarly, human CD40 can alsobe assayed in biological fluids by a competition immunoassay utilizingrhCD40 standards labeled with a detectable substance and an unlabeledanti-human CD40 antibody.

The antibodies and antibody portions of the invention preferably arecapable of neutralizing human CD40 activity both in vitro and in vivo.Accordingly, such antibodies and antibody portions of the invention canbe used to inhibit hCD40 activity, e.g., in a cell culture containinghCD40, in human subjects or in other mammalian subjects having CD40 withwhich an antibody of the invention cross-reacts. In one embodiment, theinvention provides a method for inhibiting hCD40 activity comprisingcontacting hCD40 with an antibody or antibody portion of the inventionsuch that hCD40 activity is inhibited. For example, in a cell culturecontaining, or suspected of containing hCD40, an antibody or antibodyportion of the invention can be added to the culture medium to inhibithCD40 activity in the culture.

In another embodiment, the invention provides a method for reducinghCD40 activity in a subject, advantageously from a subject sufferingfrom a disease or disorder in which CD40 activity is detrimental. Theinvention provides methods for reducing CD40 activity in a subjectsuffering from such a disease or disorder, which method comprisesadministering to the subject an antibody or antibody portion of theinvention such that CD40 activity in the subject is reduced. Preferably,the CD40 is human CD40, and the subject is a human subject.Alternatively, the subject can be a mammal expressing a CD40 to which anantibody of the invention is capable of binding. Still further thesubject can be a mammal into which CD40 has been introduced (e.g., byadministration of CD40 or by expression of a CD40 transgene). Anantibody of the invention can be administered to a human subject fortherapeutic purposes. Moreover, an antibody of the invention can beadministered to a non-human mammal expressing a CD40 with which theantibody is capable of binding for veterinary purposes or as an animalmodel of human disease. Regarding the latter, such animal models may beuseful for evaluating the therapeutic efficacy of antibodies of theinvention (e.g., testing of dosages and time courses of administration).

As used herein, the term “a disorder in which CD40 activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of CD40 in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which CD40activity is detrimental is a disorder in which reduction of CD40activity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of CD40 in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofCD40 in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, for example, using an anti-CD40 antibody as describedabove. Non-limiting examples of disorders that can be treated with theantibodies of the invention, and variants thereof, or antigen bindingfragments thereof, include those disorders discussed in the sectionbelow pertaining to pharmaceutical compositions of the antibodies of theinvention. For example, suitable disorders include, but are not limitedto, systemic lupus erythematosus (SLE), discoid lupus, lupus nephritis,sarcoidosis, juvenile arthritis, rheumatoid arthritis, psoriaticarthritis. Reiter's syndrome, ankylosing spondylitis, gouty arthritis,rejection of an organ or tissue transplant, graft versus host disease,multiple sclerosis, hyper IgE syndrome, polyarteritis nodosa, primarybiliary cirrhosis, inflammatory bowel disease. Crohn's disease, celiac'sdisease (gluten-sensitive enteropathy), autoimmune hepatitis, perniciousanemia, autoimmune hemolytic anemia, psoriasis, scleroderma, myastheniagravis, autoimmune thrombocytopenic purpura, autoimmune thyroiditis,Grave's disease, Hashimoto's thyroiditis, immune complex disease,chronic fatigue immune dysfunction syndrome (CFIDS), polymyositis anddermatomyositis, cryoglobulinemia, thrombolysis, cardiomyopathy,pemphigus vulgaris, pulmonary interstitial fibrosis, sarcoidosis, Type Iand Type II diabetes mellitus, type 1, 2, 3, and 4 delayed-typehypersensitivity, allergy or allergic disorders, asthma. Churg-Strausssyndrome (allergic granulomatosis), atopic dermatitis, allergic andirritant contact dermatitis, urtecaria, IgE-mediated allergy,atherosclerosis, vasculitis, idiopathic inflammatory myopathies,hemolytic disease. Alzheimer's disease, and chronic inflammatorydemyelinating polyneuropathy. In a particular embodiment, the disease ordisorder is a chronic inflammatory disorder. In a particular embodiment,the disorder in which CD40 activity is detrimental is an inflammatorybowel disease (IBD), including, but not limited to, Crohn's disease orulcertative colitis.

In other embodiments, the anti-CD40 antibody, or antigen bindingportion, of the invention is used to treat a disorder in which TNFαactivity is detrimental, including, but not limited to, rheumatoidarthritis, ulcerative colitis, hidradenitis suppurativa, juvenileidiopathic arthritis, psoriatic arthritis, psoriasis, ankylosingspondylitis, and Crohn's disease. In one embodiment, the disorder inwhich TNFα activity is detrimental is uveitis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat a human subjecthaving an inflammatory bowel disease (IBD).

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat ulcerativecolitis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat Crohn's disease.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat systemic lupuserythematosus (SLE).

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat sarcoidosis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention. e.g. Ab102, is used to treat a human subjecthaving juvenile arthritis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention. e.g. Ab102, is used to treat rheumatoidarthritis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention. e.g. Ab102, is used to treat psoriaticarthritis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat a human subjecthaving ankylosing spondylitis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat hidradenitissuppurativa.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat uveitis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat Sjogren's.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat psoriasis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat atopicdermatitis.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used to treat scleroderma.

Anti-CD40 mAbs of the present invention will have the potential to treatboth biologic naïve patients and anti-TNF inadequate responderpopulations due to the central role of CD40 in both innate and adaptiveimmune responses. The present invention provides a treatment capable ofinhibiting CD40 signaling suppresses molecular pathways such as TNF andIL-23 production and adhesion/co-stimulatory molecule expression thatmaintain chronic inflammation in the gut. Based on expression profilingof anti-TNF treated Crohn's patients, treatment with anti-CD40 may havethe potential to extend beyond the anti-TNF responder population totreat a broader segment of Crohn's patients. In certain embodiments, theinvention provides a method of treating a subpopulation of IBD patientswho fail to respond to anti-TNF therapy. Such IBD patients may haveCrohn's disease or ulcerative colitis and have either failed to respondto or have had a limited response to treatment with a TNFα inhibitor,such as, but not limited to, infliximab, adalimumab, certolizumab pegol,or golimumab. The anti-CD40 antagonist antibodies described herein maybe used, in to treat a TNF non-responder who has Crohn's disease orulcerative colitis. In certain embodiments, the invention includes amethod of treating a treat patient, e.g., an adult patient, withmoderately to severely active Crohn's disease who is an anti-TNFnon-responder.

Antibodies of the invention, or antigen-binding portions thereof, can beused alone or in combination to treat such diseases. It should beunderstood that the antibodies of the invention or antigen-bindingportion thereof can be used alone or in combination with an additionalagent, e.g., a therapeutic agent, said additional agent being selectedby the skilled artisan for its intended purpose. For example, theadditional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the present invention. The additional agent also can be an agent thatimparts a beneficial attribute to the therapeutic composition, e.g., anagent which affects the viscosity of the composition. It should furtherbe understood that the combinations which are to be included within thisinvention are those combinations useful for their intended purpose. Theagents set forth below are illustrative for purposes and not intended tobe limited. The combinations, which are part of this invention, can bethe antibodies of the present invention and at least one additionalagent selected from the lists below. The combination can also includemore than one additional agent, e.g., two or three additional agents ifthe combination is such that the formed composition can perform itsintended function.

The combination therapy can include one or more CD40 antagonists. e.g.,anti-CD40 antibodies or fragments thereof, formulated with, and/orco-administered with, one or more additional therapeutic agents, e.g.,one or more cytokine and growth factor inhibitors, immunosuppressants,anti-inflammatory agents (e.g., systemic anti-inflammatory agents),anti-fibrotic agents, metabolic inhibitors, enzyme inhibitors, and/orcytotoxic or cytostatic agents, mitotic inhibitors, antitumorantibiotics, immunomodulating agents, vectors for gene therapy,alkylating agents, antiangiogenic agents, antimetabolites,boron-containing agents, chemoprotective agents, hormones, antihormoneagents, corticosteroids, photoactive therapeutic agents,oligonucleotides, radionuclide agents, topoisomerase inhibitors,tyrosine kinase inhibitors, or radiosensitizers, as described in moreherein.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention. e.g. Ab102, is used in combination with asecond agent to treat an inflammatory bowel disease (IBD). In certainembodiments, the second agent is mesalamine, balsalazide, azathioprine,6-MP, methotrexate, infliximab, cenolizumab, adalimumab, golimumab,natalizumab, vedolizumab, ustekinumab, or combinations thereof.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used in combination with asecond agent to treat SLE, where the second agent isnitropaste/nitroglycerin, nifedipine, sildenafil, tadalifil, orcombinations thereof.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used in combination with asecond agent to treat SLE, where the second agent is a corticosteroid,an endogenous steroid producer, an NSAID, an anti-inflammatory agent, adisease-modifying antirheumatic drug (DMARD), an immunosuppressiveagent, an anti-coagulant, or combinations thereof.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention. e.g. Ab102, is used in combination with acorticosteroid to treat SLE. Examples of corticosteroids that may beused include prednisolone, methylprednisolone, prednisone, orcombinations thereof.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used in combination with anagent that effects endogenous steroid production, e.g., corticotropin(Acthar), to treat SLE.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used in combination with atopical or injected therapy to treat SLE. An example of such a therapyincludes, but is not limited to, cortisone, hydrocortisone, pimecrolimuscream, tacrolimus ointment, imiquimod, or combinations thereof.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used in combination with anonsteroidal anti-inflammatory drug (NSAID) to treat SLE. An example ofan NSAID that may be used in a combination therapy includes, but is notlimited to, indomethacin, nabumetone, celecoxib, ibuprofen, naproxen,diclofenac, or combinations thereof.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention. e.g. Ab102, is used in combination with ananti-inflammatory drug to treat SLE. In further embodiments, theanti-inflammatory drug is acetaminophen and/or a salicylates.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used in combination with adisease-modifying antirheumatic drugs (DMARD) to treat SLE. An exampleof a DMARD includes, but is not limited to, hydroxychloroquine(Plaquenil), chloroquine, methotrexate (Rheumatrex), leflunomide(Arava), sulfasalazine, or combinations thereof.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used in combination withBelimumab (Benlysta), Rituximab (Rituxan), intravenous Ig, orcombinations thereof.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention, e.g. Ab102, is used in combination with animmunosuppressive agent to treat SLE. Examples of an immunosuppressiveagent include, but are not limited to, azathioprine (Imuran),cyclophosphamide (Cytoxan), cyclosporine, tacrolimus, and mycophenolate.

In one embodiment, an anti-CD40 antibody, or antigen-binding portionthereof, of the invention. e.g. Ab102, is used in combination with ananti-coagulant agent to treat SLE. Examples of an anticoagulant agentinclude, but are not limited to, aspirin, heparin, warfarin, andenoxaparin (Lovenox).

Further examples of preferred additional therapeutic agents that can beco-administered and/or formulated with one or more CD40 antagonists,e.g., anti-CD40 antibodies or fragments thereof. Such combinations canbe used to treat CD40 related disorders as set forth herein. Additionalexamples of therapeutic agents that can be co-administered and/orformulated with one or more anti-CD40 antibodies or fragments thereofinclude one or more of: TNF antagonists (e.g., a soluble fragment of aTNF receptor, e.g., p55 or p75 human TNF receptor or derivativesthereof, e.g., 75 kD TNFR-IgG (75 kD TNF receptor-IgG fusion protein,ENBREL)); TNF enzyme antagonists, e.g., TNF converting enzyme (TACE)inhibitors: muscarinic receptor antagonists: TGF-beta antagonists;interferon gamma; perfenidone: chemotherapeutic agents, e.g.,methotrexate, leflunomide, or a sirolimus (rapamycin) or an analogthereof. e.g., CCI-779; COX2 and cPLA2 inhibitors; nonsteroidalanti-inflammatory drugs (NSAIDs); immunomodulators; p38 inhibitors,TPL-2, MK-2 and NFkB inhibitors, among others.

Other preferred combinations are cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-31, interferons, EMAP-II,GM-CSF, FGF, EGF, PDGF, and endothelin-1, as well as the receptors ofthese cytokines and growth factors. Antibodies of the invention, orantigen-binding portions thereof, can be combined with antibodies tocell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligandsincluding CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at differentpoints in the inflammatory cascade; preferred examples include TNFantagonists like chimeric, humanized or human TNF antibodies,adalimumab, (HUMIRA; D2E7; PCT Publication No. WO 97/29131), CA2(REMICADE), CDP 571, and soluble p55 or p75 TNF receptors, derivatives,thereof, (p75TNFRIgG (ENBREL) or p55TNFRIgG (LENERCEPT), and also TNFconverting enzyme (TACE) inhibitors; similarly IL-1 inhibitors(Interleukin-1-converting enzyme inhibitors, IL-IRA etc.) may beeffective for the same reason. Other preferred combinations includeInterleukin 4.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

In another aspect, this application features a method of treating (e.g.,curing, suppressing, ameliorating, delaying or preventing the onset of,or preventing recurrence or relapse of) or preventing a disorder inwhich CD40 activity is detrimental, in a subject. The method includes:administering to the subject a CD40 binding agent (particularly anantagonist). e.g., an anti-CD40 antibody or fragment thereof asdescribed herein, in an amount sufficient to treat or prevent theCD40-associated disorder. The CD40 antagonist. e.g., the anti-CD40antibody or fragment thereof, can be administered to the subject, aloneor in combination with other therapeutic modalities as described herein.

In another aspect, this application provides a method for detecting thepresence of CD40 in a sample in vitro (e.g., a biological sample, suchas serum, plasma, tissue, biopsy). The subject method can be used todiagnose a disorder, e.g., an inflammatory disorder. The methodincludes: (i) contacting the sample or a control sample with theanti-CD40 antibody or fragment thereof as described herein; and (ii)detecting formation of a complex between the anti-CD40 antibody orfragment thereof, and the sample or the control sample, wherein astatistically significant change in the formation of the complex in thesample relative to the control sample is indicative of the presence ofCD40 in the sample.

In yet another aspect, this application provides a method for detectingthe presence of CD40 in vivo (e.g., in vivo imaging in a subject). Thesubject method can be used to diagnose a disorder, e.g., aCD40-associated disorder. The method includes: (i) administering theanti-CD40 antibody or fragment thereof as described herein to a subjector a control subject under conditions that allow binding of the antibodyor fragment to CD40; and (ii) detecting formation of a complex betweenthe antibody or fragment and CD40, wherein a statistically significantchange in the formation of the complex in the subject relative to thecontrol subject is indicative of the presence of CD40.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are known in the art (Winter, et al. U.S. Pat. Nos.5,648,260; 5,624,821). The Fc portion of an antibody mediates severalimportant effector functions e.g. cytokine induction, ADCC,phagocytosis, complement dependent cytotoxicity (CDC) andhalf-life/clearance rate of antibody and antigen-antibody complexes. Insome cases these effector functions are desirable for therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRsand complement C1q, respectively. Neonatal Fc receptors (FcRn) are thecritical components determining the circulating half-life of antibodies.In still another embodiment at least one amino acid residue is replacedin the constant region of the antibody, for example the Fc region of theantibody, such that effector functions of the antibody are altered.

One embodiment provides a labeled antibody, or an antigen-bindingportion thereof, wherein an antibody or antibody portion of theinvention is derivatized or linked to one or more functional molecule(s)(e.g., another peptide or protein). For example, a labeled antibody, oran antigen-binding portion thereof, of the invention can be derived byfunctionally linking an antibody or antibody portion of the invention(by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherantibody (e.g., a bispecific antibody or a diabody), a detectable agent,a pharmaceutical agent, a protein or peptide that can mediate theassociation of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag), and/or acytotoxic or therapeutic agent selected from the group consisting of amitotic inhibitor, an antitumor antibiotic, an immunomodulating agent, avector for gene therapy, an alkylating agent, an antiangiogenic agent,an antimetabolite, a boron-containing agent, a chemoprotective agent, ahormone, an antihormone agent, a corticosteroid, a photoactivetherapeutic agent, an oligonucleotide, a radionuclide agent, atopoisomerase inhibitor, a tyrosine kinase inhibitor, a radiosensitizer,and a combination thereof.

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine. 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

Another embodiment of the invention provides a crystallized antibody, oran antigen-binding portion thereof. Preferably the invention relates tocrystals of whole anti-CD40 antibodies and fragments thereof asdisclosed herein, and formulations and compositions comprising suchcrystals. In one embodiment the crystallized antibody, or anantigen-binding portion thereof, has a greater half-life in vivo thanthe soluble counterpart of the antibody, or an antigen-binding portionthereof. In another embodiment the antibody, or an antigen-bindingportion thereof, retains biological activity after crystallization.

Crystallized antibody, or an antigen-binding portion thereof, of theinvention may be produced according methods known in the art and asdisclosed in WO 02072636, incorporated herein by reference.

IV. Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising anantibody, or antigen-binding portion thereof, of the invention and apharmaceutically acceptable carrier.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody, or antibody portion, are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

The pharmaceutical compositions comprising antibodies of the inventionare for use in, but not limited to, diagnosing, detecting, or monitoringa disorder, in preventing, treating, managing, or ameliorating of adisorder or one or more symptoms thereof, and/or in research. In aspecific embodiment, a composition comprises one or more antibodies ofthe invention. In another embodiment, the pharmaceutical compositioncomprises one or more antibodies of the invention and one or moreprophylactic or therapeutic agents other than antibodies of theinvention for treating a disorder in which CD40 activity is detrimental.Preferably, the prophylactic or therapeutic agents known to be usefulfor or having been or currently being used in the prevention, treatment,management, or amelioration of a disorder or one or more symptomsthereof. In accordance with these embodiments, the composition mayfurther comprise of a carrier, diluent or excipient.

The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a prophylactic or therapeutic agent of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural administration, intratumoral administration, andmucosal administration (e.g., intranasal and oral routes). In addition,pulmonary administration can be employed. e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064,5,855,913, 5,290,540, and 4,880.078; and PCT Publication Nos. WO92/19244, WO 97/32572. WO 97/44013, WO 98/31346, and WO 99/66903, eachof which is incorporated herein by reference their entireties. In oneembodiment, an antibody of the invention, combination therapy, or acomposition of the invention is administered using Alkermes AIRpulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).In a specific embodiment, prophylactic or therapeutic agents of theinvention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., TISSUEL), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention antagonists is administered locally to the affected area to asubject to prevent, treat, manage, and/or ameliorate a disorder or asymptom thereof. In another embodiment, an effective amount of one ormore antibodies of the invention is administered locally to the affectedarea in combination with an effective amount of one or more therapies(e.g., one or more prophylactic or therapeutic agents) other than anantibody of the invention of a subject to prevent, treat, manage, and/orameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent of theinvention can be delivered in a controlled release or sustained releasesystem. In one embodiment, a pump may be used to achieve controlled orsustained release (see Langer, supra: Sefton, 1987, CRC Crit. RefBiomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek etal., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymericmaterials can be used to achieve controlled or sustained release of thetherapies of the invention (see e.g., Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability. Drug Product Design and Performance.Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983,J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howardet al. 1989, J. Neurosurg. 7 1:105); U.S. Pat. Nos. 5,679,377;5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO99/15154; and PCT Publication No. WO 99/20253. Examples of polymers usedin sustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In apreferred embodiment, the polymer used in a sustained releaseformulation is inert, free of leachable impurities, stable on storage,sterile, and biodegradable. In yet another embodiment, a controlled orsustained release system can be placed in proximity of the prophylacticor therapeutic target, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human ColonCancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology39:179-189, Song et al., 1995. “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions.” PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun: Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see. e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

If the compositions of the invention are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity preferablygreater than water are typically employed. Suitable formulationsinclude, without limitation, solutions, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like, which are, ifdesired, sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, preferably in combination with a solid or liquidinert carrier, is packaged in a mixture with a pressurized volatile(e.g., a gaseous propellant, such as freon) or in a squeeze bottle.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art.

If the method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the method of the invention comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gel caps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinized maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See. e.g., U.S. Pat. Nos. 6,019,968, 5,985,320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903, each of which is incorporated herein byreference their entireties. In a specific embodiment, an antibody of theinvention, combination therapy, and/or composition of the invention isadministered using Alkermes AIR pulmonary drug delivery technology(Alkermes, Inc., Cambridge. Mass.).

The method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the invention may additionally comprise of administrationof compositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents, or pharmaceutical compositions ofthe invention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. Preferably, one or more of the prophylactic or therapeuticagents or pharmaceutical compositions of the invention is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,at least 75 mg, or at least 100 mg. The lyophilized prophylactic ortherapeutic agents or pharmaceutical compositions of the inventionshould be stored at between 2° C., and 8° C. in its original containerand the prophylactic or therapeutic agents, or pharmaceuticalcompositions of the invention should be administered within 1 week,preferably within 5 days, within 72 hours, within 48 hours, within 24hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours,or within 1 hour after being reconstituted. In an alternativeembodiment, one or more of the prophylactic or therapeutic agents orpharmaceutical compositions of the invention is supplied in liquid formin a hermetically sealed container indicating the quantity andconcentration of the agent. Preferably, the liquid form of theadministered composition is supplied in a hermetically sealed containerat least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, atleast 75 mg/ml or at least 100 mg/ml. The liquid form should be storedat between 2° C., and 8° C. in its original container.

The antibodies and antibody-portions of the invention can beincorporated into a pharmaceutical composition suitable for parenteraladministration. Preferably, the antibody or antibody-portions will beprepared as an injectable solution containing 0.1-250 mg/ml antibody.The injectable solution can be composed of either a liquid orlyophilized dosage form in a flint or amber vial, ampule or pre-filledsyringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, atpH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but arenot limited to, sodium succinate, sodium citrate, sodium phosphate orpotassium phosphate. Sodium chloride can be used to modify the toxicityof the solution at a concentration of 0-300 mM (optimally 150 mM for aliquid dosage form). Cryoprotectants can be included for a lyophilizeddosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Othersuitable cryoprotectants include trehalose and lactose. Bulking agentscan be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 2-4%). Stabilizers can be used in both liquid andlyophilized dosage forms, principally 1-50 mM L-Methionine (optimally5-10 mM). Other suitable bulking agents include glycine, arginine, canbe included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants. The pharmaceutical composition comprising theantibodies and antibody-portions of the invention prepared as aninjectable solution for parenteral administration, can further comprisean agent useful as an adjuvant, such as those used to increase theabsorption, or dispersion of a therapeutic protein (e.g., antibody). Aparticularly useful adjuvant is hyaluronidase, such as HYLENEX(recombinant human hyaluronidase). Addition of hyaluronidase in theinjectable solution improves human bioavailability following parenteraladministration, particularly subcutaneous administration. It also allowsfor greater injection site volumes (i.e. greater than 1 ml) with lesspain and discomfort, and minimum incidence of injection site reactions.(see WO2004078140, US2006104968 incorporated herein by reference).

In one embodiment, the invention includes a pharmaceutical compositioncomprising an antibody of the invention, e.g., antibody Ab102, histidineand a polysorbate, e.g., polysorbate 80. In one embodiment, theinvention includes a pharmaceutical composition comprising an antibodycomprising a heavy chain comprising the amino acids of SEQ ID NO: 41 anda light chain comprising the amino acids of SEQ ID NO: 40, histidine anda polysorbate, e.g., polysorbate 80. In certain embodiments, thepharmaceutical composition is lyophilized.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody-portions of the present invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker. Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion of the inventionmay be orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

In other embodiments, an antibody or antibody portion of the inventionmay be conjugated to a polymer-based species such that saidpolymer-based species may confer a sufficient size upon said antibody orantibody portion of the invention such that said antibody or antibodyportion of the invention benefits from the enhanced permeability andretention effect (EPR effect) (See also PCT Publication No.WO2006/042146A2 and U.S. Publication Nos. 2004/0028687A1,2009/0285757A1, and 2011/0217363A1, and U.S. Pat. No. 7,695,719 (each ofwhich is incorporated by reference herein in its entirety and for allpurposes).

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is formulated with and/or co-administered with one or moreadditional therapeutic agents that are useful for treating disorders inwhich CD40 activity is detrimental. For example, an anti-hCD40 antibodyor antibody portion of the invention may be formulated and/orco-administered with one or more additional antibodies that bind othertargets (e.g., antibodies that bind cytokines or that bind cell surfacemolecules). Furthermore, one or more antibodies of the invention may beused in combination with two or more of the foregoing therapeuticagents. Such combination therapies may advantageously utilize lowerdosages of the administered therapeutic agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.

In certain embodiments, an antibody to CD40 or fragment thereof islinked to a half-life extending vehicle known in the art. Such vehiclesinclude, but are not limited to, the Fc domain, polyethylene glycol, anddextran. Such vehicles are described, e.g., in U.S. application Ser. No.09/428,082 and published PCT Application No. WO 99/25044, which arehereby incorporated by reference for any purpose.

In a specific embodiment, nucleic acid sequences comprising nucleotidesequences encoding an antibody of the invention or another prophylacticor therapeutic agent of the invention are administered to treat,prevent, manage, or ameliorate a disorder or one or more symptomsthereof by way of gene therapy. Gene therapy refers to therapy performedby the administration to a subject of an expressed or expressiblenucleic acid. In this embodiment of the invention, the nucleic acidsproduce their encoded antibody or prophylactic or therapeutic agent ofthe invention that mediates a prophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. For general reviews of the methodsof gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95: Tolstoshev, 1993, Ann.Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, Science 260:926-932(1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217;May, 1993, TIBTECH 11(5):155-215. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons,NY (1993); and Kriegler. Gene Transfer and Expression, A LaboratoryManual, Stockton Press, NY (1990). Detailed description of variousmethods of gene therapy is provided in US20050042664 A1 which isincorporated herein by reference.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the present invention in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1: Antagonist Anti-Human CD40 (hCD40) MonoclonalAntibodies

In order to identify CD40 specific antagonist antibodies, hybridomatechnology was used to isolate murine monoclonal anti-CD40 antibodies.

Briefly, mice were immunized with human CD40 antigen and adjuvant. Afterimmunization, which included several administrations of the antigen overseveral weeks, serum from each immunized animal was collected. The serumwas then tested using standard ELISA and flow cytometry assays toidentify serum having antibodies that were able to detect CD40. Once thepresence of CD40-specific antibodies was detected in the mouse serumbased on the binding assays, the mouse spleen was harvested andantibody-producing cells were isolated according to standard techniques.The splenocytes were then fused by known techniques to formantibody-producing myeloma cells. After fusion, hybridomas were screenedby ELISA and flow cytometry to determine the various antibody CD40blocking and neutralization characteristics.

Following screening, while the majority of antibodies were identified ashaving agonist activity, three of the murine monoclonal antibodies (Ab1,Ab2 and Ab3) were identified as having antagonist activity to CD40without substantial agonist activity. The heavy and light chain aminoacid sequences of these three murine antibodies are described below inTables 7 to 9. CDRs within the variable heavy (VH) and variable light(VL) chains are shown by bold text (CDR1, CDR2, and CDR3, respectively).

TABLE 7 VH and VL amino acid sequences of murine antibody 1 (Ab1) SEQ IDClone Antibody Residue NO. name Region description Amino acid sequence 5Ab1 VH EVQLVESGGGLVKPGGSLKVSCAASGFTFSD YGMNWVRQAPEKGLEWIAYISSGRSNIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAM YYCARSWGYFDVWGTGTTVTVSS 6 Ab1 CDR-H1Residues 26-35 GFTFSDYGMN of SEQ ID NO.: 5 7 Ab1 CDR-H2 Residues 50-66YISSGRSNIYYADTVKG of SEQ ID NO.: 5 8 Ab1 CDR-H3 Residues 99-105 SWGYFDVof SEQ ID NO.: 5 9 Ab1 VL DIVMTQSPSSLTVTAGEMVTMSCKSSQSLLNSGNQKNYLTWFQQKPGQPPKLLIYWASTRES GVPDRFAGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLEIK 10 Ab1 CDR-L1 Residues 24-40 KSSQSLLNSGNQKNYLT ofSEQ ID NO.: 9 11 Ab1 CDR-L2 Residues 56-62 WASTRES of SEQ ID NO.: 9 12Ab1 CDR-L3 Residues 95-103 QNDYTYPLT of SEQ ID NO.: 9

TABLE 8 VH and VL amino acid sequences of murine antibody 3 (Ab3) SEQ IDClone Antibody Residue NO. name Region description Amino acid sequence44 Ab3 VH QVQLQQSGAELARPGASVKMSCKAFGYTFTSYTMHWVKQRPGQGLEWIGYINPSSDYPNYNQ KFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWGYSFDYWGQGTTLTVSS 45 Ab3 CDR-H1 Residues 26-35 GYTFTSYTMH of SEQID NO.: 44 46 Ab3 CDR-H2 Residues 50-66 YINPSSDYPNYNQKFKD of SEQ ID NO.:44 47 Ab3 CDR-H3 Residues 99-105 WGYSFDY of SEQ ID NO.: 44 48 Ab3 VLDIVMTQAAPSVSVIPGESVSISCRSSKSLLH SNGNTYLYWFLQRPGQSPQYLIYRMSTLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC MQHLEYPLTFGAGTKLELK 49 Ab3 CDR-L1Residues 24-39 RSSKSLLHSNGNTYLY of SEQ ID NO.: 48 50 Ab3 CDR-L2 Residues55-61 RMSTLAS of SEQ ID NO.: 48 51 Ab3 CDR-L3 Residues 94-102 MQHLEYPLTof SEQ ID NO.: 48

TABLE 9 VH and VL amino acid sequences of murine antibody 2 (Ab2) SEQ IDAntibody Residue NO. Clone name Region description Amino acid sequence75 Ab2 VH EVQLVESGGGLVKPGGSLKVSCAASGFTFSDYGMNWVRQSPEKGLEWIAYISSGRGNIYYAD TVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARSWGYFDVWGTGTTVTVSS 6 Ab2 CDR-H1 Residues 26-35 GFTFSDYGMN of SEQ IDNO.: 75 42 Ab2 CDR-H2 Residues 50-66 YISSGRGNIYYADTVKG of SEQ ID NO.: 758 Ab2 CDR-H3 Residues 99-105 SWGYFDV of SEQ ID NO.: 75 76 Ab2 VLDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLN SGNQKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYY CQNDYTYPLTFGAGTKLELK 10 Ab2 CDR-L1Residues 24-40 KSSQSLLNSGNQKNYLT of SEQ ID NO.: 76 11 Ab2 CDR-L2Residues 56-62 WASTRES of SEQ ID NO.: 76 12 Ab2 CDR-L3 Residues 95-103QNDYTYPLT of SEQ ID NO.: 76

Consensus sequences of the CDR regions from the three anti-CD40antagonist murine monoclonal antibodies (Ab1, Ab2 and Ab3) wereidentified and are provided above in Table 5. Alignments of the variableregion amino acid sequences of the three murine antibodies are alsoprovided in FIGS. 4A (light chain) and 4B (heavy chain).

The murine heavy and light chain variable regions (VH and VL) of thethree antibodies were cloned using reverse transcriptase-PCR (RT-PCR).These VH and VL regions (described above in Tables 7 to 9) weresubsequently cloned into vectors comprising human immunoglobulin (Ig)constant regions, and then expressed in mammalian host cells as chimericantibodies. These human chimeric antibodies (human constant and murinevariable regions) were then characterized using in vitro assays todetermine whether they each had an antagonist and/or agonist effect.

FACS analysis was used to determine whether the three chimeric anti-CD40antibodies could bind either huCD40 or cyno CD40 expressed on HumanEmbryonic Kidney (HEK) cells. While each of the three chimericantibodies could bind human CD40 expressed on HEK cells, only two of thechimeric antibodies recognized cyno—chimeric Antibody 3 (chAb3) did notbind to cyno CD40. The results of the FACS binding study are summarizedin Table 10.

FACS analysis was also used to determine whether the three chimericantibodies could inhibit binding of soluble CD40 ligand (sCD40L) toCD40. Using CD40 expressing HEK cells, IC50 values were measured. Asdescribed in Table 10, each of the chimeric antibodies was able to blockCD40 binding to its ligand.

In addition to binding assays, antagonist and agonist activities weremeasured using a CD40-expressing reporter cell line expressing humanCD40 linked to NFkB mediated alkaline phosphatase (AP). In theCD40-expressing reporter cell line assay, when signal is receivedthrough CD40, NFkB activation leads to secretion of AP which is measuredby colorimetric substrate. To determine antagonist activity, the CD40reporter cell line (HEK) was cultured with either with Jurkat cell lineexpressing CD40L (to provide physiological ligand interaction) or withsoluble CD40L (e.g., His-CD40L referenced in Table 10). The ability ofanti-CD40 antibodies to block the NFkB signal was measured. To measureagonist activity, human CD40 reporter cell line was directly treatedwith anti-CD40 antibodies and NFkB signal was measured. RepresentativeCD40 antagonist and agonist assay data for the three chimeric antibodiesare summarized in Table 10 and FIGS. 1A and 1B.

TABLE 10 Summary of Functional Characteristics of anti-CD40 ChimericAntibodies HEK Agonist: Antagonist: Chimeric HEK HEK CD40 HEK His-CD40LAntagonist: CD40 huCD40 cyCD40 FACS huCD40 reporter Jurkat/ AntibodiesFACS FACS Blocking reporter assay Reporter assay (hCg1-LALA) bindingbinding IC50 nM assay IC50 nM IC50 nM chimeric Ab1 Yes Yes 2.3 No 0.4 51(chAb1) chimeric Ab3 Yes No 1.4 No 0.2 0.9 (chAb3) chimeric Ab2 Yes Yes1.4 No 0.3 3.4 (chAb2)

As described in Table 10 and FIG. 1, the anti-CD40 chimeric antibodiesshowed antagonist activity with no detectable agonist activity. Based onthe results from the above experiments, the heavy and light chainvariable regions from anti-CD40 antagonist antibodies Ab1, and Ab3 wereselected for humanization.

Example 2: Humanization of Antagonist Anti-CD40 Antibodies Ab1 and Ab3Humanization of Antagonist Anti-CD40 Antibody Ab1

Humanized antibodies were generated based on the variable heavy (VH) andvariable light (VL) CDR sequences of Ab1. Specifically, human germlinesequences were selected for constructing CDR-grafted, humanized Ab1antibodies, where the CDR domains of the VH and VL chains of Ab1 wasgrafted onto different human heavy and light chain acceptor sequences.Based on the alignments with the VH and VL sequences of monoclonalantibody Ab1, the following human sequences were selected as acceptors:

1. IGHV3-21*01 and IGHJ6*01 for constructing heavy chain acceptorsequences2. IGHV3-48*01 and IGHJ6*01 as an alternative acceptor for constructingheavy chain3. IGKV4-1*01 and IGKJ2*01 for constructing light chain acceptorsequences4. IGKV2-40*01 and IGKJ2*01 as an alternative acceptor for constructinglight chainCDR-grafted antibodies were then prepared by grafting the correspondingVH and VL CDRs of Ab1 into the acceptor sequences described in 1-4above.

To generate a humanized antibody with framework backmutation(s),framework mutations were identified and introduced into the CDR-graftedantibodies. These mutations were introduced using standard techniques,including de novo synthesis of the variable domain with thebackmutation(s) and mutagenic oligonucleotide primers in polymerasechain reactions. Different combinations of back mutations and othermutations were constructed for each of the CDR-grafted antibodies(containing the CDRs of antibody Ab1) as follows. (Note: Residue numbersfor the below-mentioned mutations are based on the Kabat numberingsystem.)

For the heavy chains of the CDR-grafted antibodies, one or more of thefollowing Vernier and VH/VL interfacing residues were back mutated: V48Iand/or S49A.

For light chains of the CDR-grafted antibodies, the following Vernierand VH/VL interfacing residue was back mutated: Y36F.

Descriptions of the variable regions of the humanized antibodies derivedfrom murine monoclonal Ab1 are provided below:

-   -   Humanized Ab1 (huAb1VH.1) is a CDR-grafted Ab1 VH containing        IGHV3-21*01 and IGHJ6*01 framework sequences;    -   Humanized Ab1VH.1a (huAb1VH.1A) is a humanized heavy chain        comprising the amino acid sequences of huAb1VH.1 with the        following two framework backmutations: V48I, S49A;    -   Humanized Ab1 VL.1 (huAb1VL.1) is a CDR-grafted Ab1 VL        containing IGKV4-1*01 and IGKJ2*01 framework sequences; and    -   Humanized Ab1 VL.1a (huAb1VL.1A) is a humanized light chain        based on huAb1VL.1 and contains 1 proposed framework        back-mutations: Y36F.        Note: IGHV3-21_IGHJ6 refers to an antibody comprising variable        sequences corresponding to IGHV3-21*01 and IGHJ6*01.

The humanized variable regions were then cloned into IgG expressionvectors for functional characterization of four different humanizedantibodies based on the following heavy and light chain variable regioncombinations:

A. huAb1VH.1/VL.1B. huAb1VH.1A/VL.1C. huAb1VH.1/VL.1AD. huAb1VH.1A/VL.1A

The variable region and CDR amino acid sequences of the foregoinghumanized antibodies are described in Table 11 below.

TABLE 11 VH and VL sequences of humanized versions of amibody 1 (huAb1)SEQ ID Antibody NO: Clone Region Residues Amino acid sequence 13huAb1VH.1/VL.1 VH VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWVSYISSGRSNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 6 huAb1VH.1/VL.1 CDR-H1 Residues 26-35GFTFSDYGMN of SEQ ID NO.: 13 7 huAb1VH.1/VL.1 CDR-H2 Residues 50-66YISSGRSNIYYADTVKG of SEQ ID NO.: 13 8 huAb1VH.1/VL.1 CDR-H3 Residues99-105 SWGYFDV of SEQ ID NO.: 13 14 huAb1VH.1/VL.1 VL VLDIVMTQSPDSLAVSLGERATINCKSSQSLL NSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQNDYTYPLTFGQGTKLEIK 10 huAb1VH.1/VL.1CDR-L1 Residues 24-40 KSSQSLLNSGNQKNYLT of SEQ ID NO.: 14 11huAb1VH.1/VL.1 CDR-L2 Residues 56-62 WASTRES of SEQ ID NO.: 14 12huAb1VH.1/VL.1 CDR-L3 Residues 95-103 QNDYTYPLT of SEQ ID NO.: 14 15huAb1VH.1A/VL.1 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRSNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 6 huAb1VH.1A/VL.1 CDR-H1 Residues 26-35GFTFSDYGMN of SEQ ID NO.: 15 7 huAb15VH.1A/VL.1 CDR-H2 Residues 50-66YISSGRSNIYYADTVKG of SEQ ID NO.: 15 8 huAb1VH.1A/VL.1 CDR-H3 Residues99-105 SWGYFDV of SEQ ID NO.: 15 14 huAb1VH.1A/VL.1 VLDIVMTQSPDSLAVSLGERATINCKSSQSLL NSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQNDYTYPLTFGQGTKLEIK 10huAb1VH.1A/VL.1 CDR-L1 Residues 24-40 KSSQSLLNSGNQKNYLT of SEQ ID NO.:14 11 huAb1VH.1A/VL.1 CDR-L2 Residues 56-62 WASTRES of SEQ ID NO.: 14 12huAb1VH.1A/VL.1 CDR-L3 Residues 95-103 QNDYTYPLT of SEQ ID NO.: 14 13huAb1VH.1/VL.1A VH VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWVSYISSGRSNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 6 huAb1VH.1/VL.1A CDR-H1 Residues 26-35GFTFSDYGMN of SEQ ID NO.: 13 7 huAb1VH.1/VL.1A CDR-H2 Residues 50-66YISSGRSNIYYADTVKG of SEQ ID NO.: 13 8 huAb1VH.1/VL.1A CDR-H3 Residues99-105 SWGYFDV of SEQ ID NO.: 13 16 huAb1VH.1/VL.1A VL VLDIVMTQSPDSLAVSLGERATINCKSSQSLL NSGNQKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQNDYTYPLTFGQGTKLEIK 10huAb1VH.1/VL.1A CDR-L1 Residues 24-40 KSSQSLLNSGNQKNYLT of SEQ ID NO.:16 11 huAb1VH.1/VL.1A CDR-L2 Residues 56-62 WASTRES of SEQ ID NO.: 16 12huAb1VH.1/VL.1A CDR-L3 Residues 95-103 QNDYTYPLT of SEQ ID NO.: 16 15huAb1VH.1A/VL.1A VH VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRSNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 6 huAb1VH.1A/VL.1A CDR-H1 Residues 26-35GFTFSDYGMN of SEQ ID NO.: 15 7 huAb1VH.1A/VL.1A CDR-H2 Residues 50-66YISSGRSNIYYADTVKG of SEQ ID NO.: 15 8 huAb1VH.1A/VL.1A CDR-H3 Residues99-105 SWGYFDV of SEQ ID NO.: 15 16 huAb1VH.1A/VL.1A VL VLDIVMTQSPDSLAVSLGERATINCKSSQSLL NSGNQKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQNDYTYPLTFGQGTKLEIK 10huAb1VH.1A/VL.1A CDR-L1 Residues 24-40 KSSQSLLNSGNQKNYLT of SEQ ID NO.:16 11 huAb1VH.1A/VL.1A CDR-L2 Residues 56-62 WASTRES of SEQ ID NO.: 1612 huAb1VH.1A/VL.1A CDR-L3 Residues 95-103 QNDYTYPLT of SEQ ID NO.: 16As described above, the CDRs of the humanized versions of the VH and VLregions of Ab1 were identical to the murine Ab1 antibody.

Humanization of Antagonist Anti-CD40 Antibody 3 (Ab3)

Humanized antibodies were also generated based on the variable heavy(VH) and variable light (VL) CDR sequences of Ab3. Human germlinesequences were selected for constructing CDR-grafted, humanized Ab3antibodies, where the CDR domains of the VH and VL chains of Ab3 weregrafted onto different human heavy and light chain acceptor sequences.Based on the alignments with the VH and VL sequences of monoclonalantibody Ab3, the following human sequences were selected as acceptors:

1. IGHV3-69*06 and IGHJ6*01 for constructing heavy chain acceptorsequences2. IGHV1-18*01 and IGHJ6*01 as an alternative acceptor for constructingheavy chain3. IGKV2-29*02 and IGKJ2*01 for constructing light chain acceptorsequences4. IGKV2-28*01 and IGKJ2*01 as an alternative acceptor for constructinglight chainCDR-grafted antibodies were prepared by grafting the corresponding VHand VL CDRs of Ab3 into the acceptor sequences described in 1-4 above.

To generate humanized antibody with framework backmutation(s), a numberof framework mutations were identified and introduced into theCDR-grafted antibodies. These mutations were introduced using standardtechniques, including de novo synthesis of the variable domain with thebackmutation(s) and mutagenic oligonucleotide primers in polymerasechain reactions. Different combinations of mutations, including backmutations, were constructed for each of the CDR-grafted antibodies(containing the CDRs of antibody Ab3). (Note: Residue numbers for thebelow-mentioned mutations are based on the Kabat numbering system.).

For heavy chains Ab3, one or more of the following Vernier and VH/VLinterfacing residues were back mutated: M48I, V67A, 169L. In addition,changes to Q1E were considered. The Q1E mutation was introduced in orderto prevent pyroglutamate formation.

For light chains Ab3, one or more of the following Vernier and VH/VLinterfacing residues were back mutated: Y36F, L46Y.

Descriptions of the variable regions of the humanized antibodies derivedfrom murine monoclonal Ab3 are described below:

-   -   huAb3VH.1z is a CDR-grafted, humanized Ab3 VH containing        IGHV1-69*06 and IGHJ6*01 framework sequences.    -   huAb3VH.1 is based on huAb3VH.1z with a Q1E change to prevent        pyroglutamate formation    -   huAb3VH.1A is a humanized design based on huAb3VH.1 and contains        3 additional framework back-mutations: M48I, V67A, 169L.    -   huAb3VH.1b is an intermediate design between huAb3VH.1 and        huAb3VH.1A and contains 1 proposed framework back-mutation:        169L.    -   huAb3VL.1 is a CDR-grafted, humanized Ab3 VL containing        IGKV2-28*01 and IGKJ2*01 framework sequences.    -   huAb3VL. A is a humanized design based on huAb3VL.1 and contains        2 framework back-mutations: Y36F, L46Y.    -   huAb3VL. B is an intermediate design between huAb3VL.1 and        huAb3VL. A. It contains 1 proposed framework back-mutation:        L46Y.        Note also that * IGHV1-69_IGHJ6 is made up of IGHV1-69*06 and        IGHJ6*01 germline sequences.

The humanized variable regions were then cloned into IgG expressionvectors for functional characterization of nine different humanizedantibodies based on the following combinations of heavy and light chainvariable regions:

A. huAb3VH.1/VL.1B. huAb3VH.1B/VL.1C. huAb3VH.1A/VL.1D. huAb3VH.1/VL.1AE. huAb3VH.1B/VL.1AF. huAb3VH.1A/VL.1AG. huAb3VH.1/VL.1BH. huAb3VH.1B/VL.1BI. huAb3VH.1A/VL.1BThe variable region and CDR amino acid sequences of the foregoinghumanized antibodies are described in Table 12 below.

TABLE 12 Amino acid sequences of VH and VL regions of humanized Ab3antibodies SEQ ID Antibody NO: Clone Region Residues Amino acid sequence52 huAb3VH.1/VL.1 VH EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMHWVRQAPGQGLEWMGYINPSSDYPNY NQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDYWGQGTTVTVSS 45 huAb3VH.1/VL.1 CDR-H1 Residues 26-35GYTFTSYTMH of SEQ ID NO.: 52 46 huAb3VH.1/VL.1 CDR-H2 Residues 50-66YINPSSDYPNYNQKFKD of SEQ ID NO.: 52 47 huAb3VH.1/VL.1 CDR-H3 Residues99-105 WGYSFDY of SEQ ID NO.: 52 53 huAb3VH.1/VL.1 VLDIVMTQSPLSLPVTPGEPASISCRSSKSLL HSNGNTYLYWYLQKPGQSPQLLIYRMSTLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPLTFGQGTKLEIK 49 huAb3VH.1/VL.1CDR-L1 Residues 24-39 RSSKSLLHSNGNTYLY of SEQ ID NO.: 53 50huAb3VH.1/VL.1 CDR-L2 Residues 55-61 RMSTLAS of SEQ ID NO.: 53 51huAb3VH.1/VL.1 CDR-L3 Residues 94-102 MQHLEYPLT of SEQ ID NO.: 53 54huAb3VH.1B/VL.1 VH EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMHWVRQAPGQGLEWMGYINPSSDYPNY NQKFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDYWGQGTTVTVSS 45 huAb3VH.1B/VL.1 CDR-H1 Residues 26-35GYTFTSYTMH of SEQ ID NO.: 54 46 huAb3VH.1B/VL.1 CDR-H2 Residues 50-66YINPSSDYPNYNQKFKD of SEQ ID NO.: 54 47 huAb3VH.1B/VL.1 CDR-H3 Residues99-105 WGYSFDY of SEQ ID NO.: 54 53 huAb3VH.1B/VL.1 VLDIVMTQSPLSLPVTPGEPASISCRSSKSLL HSNGNTYLYWYLQKPGQSPQLLIYRMSTLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPLTFGQGTKLEIK 49 huAb3VH.1B/VL.1CDR-L1 Residues 24-39 RSSKSLLHSNGNTYLY of SEQ ID NO.: 53 50huAb3VH.1B/VL.1 CDR-L2 Residues 55-61 RMSTLAS of SEQ ID NO.: 53 51huAb3VH.1B/VL.1 CDR-L3 Residues 94-102 MQHLEYPLT of SEQ ID NO.: 53 55huAb3VH.1A/VL.1 VH EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMHWVRQAPGQGLEWIGYINPSSDYPNY NQKFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDYWGQGTTVTVSS 45 huAb3VH.1A/VL.1 CDR-H1 Residues 26-35GYTFTSYTMH of SEQ ID NO.: 55 46 huAb3VH.1A/VL.1 CDR-H2 Residues 50-66YINPSSDYPNYNQKFKD of SEQ ID NO.: 55 47 huAb3VH.1A/VL.1 CDR-H3 Residues99-105 WGYSFDY of SEQ ID NO.: 55 53 huAb3VH.1A/VL.1 VLDIVMTQSPLSLPVTPGEPASISCRSSKSLL HSNGNTYLYWYLQKPGQSPQYLIYRMSTLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPLTFGQGTKLEIK 49 huAb3VH.1A/VL.1CDR-L1 Residues 24-39 RSSKSLLHSNGNTYLY of SEQ ID NO.: 53 50huAb3VH.1A/VL.1 CDR-L2 Residues 55-61 RMSTLAS of SEQ ID NO.: 53 51huAb3VH.1A/VL.1 CDR-L3 Residues 94-102 MQHLEYPLT of SEQ ID NO.: 53 52huAb3VH.1/VL.1A VL EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMHWVRQAPGQGLEWMGYINPSSDYPNY NQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDYWGQGTTVTVSS 45 huAb3VH.1/VL. CDR-H1 Residues 26-35GYTFTSYTMH 1A of SEQ ID NO.: 52 46 huAb3VH.1/VL. CDR-H2 Residues 50-66YINPSSDYPNYNQKFKD 1A of SEQ ID NO.: 52 47 huAb3VH.1/VL. CDR-H3 Residues99-105 WGYSFDY 1A of SEQ ID NO.: 52 56 huAb3VH.1/VL.1A VLDIVMTQSPLSLPVTPGEPASISCRSSKSLL HSNGNTYLYWFLQKPGQSPQYLIYRMSTLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPLTFGQGTKLEIK 49 huAb3VH.1/VL.CDR-L1 Residues 24-39 RSSKSLLHSNGNTYLY 1A of SEQ ID NO.: 56 50huAb3VH.1/VL. CDR-L2 Residues 55-61 RMSTLAS 1A of SEQ ID NO.: 56 51huAb3VH.1/VL. CDR-L3 Residues 94-102 MQHLEYPLT 1A of SEQ ID NO.: 56 54huAb3VH.1B/VL.1A VH EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMHWVRQAPGQGLEWMGYINPSSDYPNY NQKFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDYWGQGTTVTVSS 45 huAb3VH.1B/VL.1A CDR-H1 Residues 26-35GYTFTSYTMH of SEQ ID NO.: 54 46 huAb3VH.1B/VL.1A CDR-H2 Residues 50-66YINPSSDYPNYNQKFKD of SEQ ID NO.: 54 47 huAb3VH.1B/VL.1A CDR-H3 Residues99-105 WGYSFDY of SEQ ID NO.: 54 56 huAb3VH.1B/VL.1A VLDIVMTQSPLSLPVTPGEPASISCRSSKSLL HSNGNTYLYWFLQKPGQSPQYLIYRMSTLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPLTFGQGTKLEIK 49huAb3VH.1B/VL.1A CDR-L1 Residues 24-39 RSSKSLLHSNGNTYLY of SEQ ID NO.:56 50 huAb3VH.1B/VL.1A CDR-L2 Residues 55-61 RMSTLAS of SEQ ID NO.: 5651 huAb3VH.1B/VL.1A CDR-L3 Residues 94-102 MQHLEYPLT of SEQ ID NO.: 5655 huAb3VH.1A/VL.1A VH EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMHWVRQAPGQGLEWIGYINPSSDYPNY NQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDYWGQGTTVTVSS 45 huAb3VH.1A/VL.1A CDR-H1 Residues 26-35GYTFTSYTMH of SEQ ID NO.: 55 46 huAb3VH.1A/VL.1A CDR-H2 Residues 50-66YINPSSDYPNYNQKFKD of SEQ ID NO.: 55 47 huAb3VH.1A/VL.1A CDR-H3 Residues99-105 WGYSFDY of SEQ ID NO.: 55 56 huAb3VH.1A/VL.1A VLDIVMTQSPLSLPVTPGEPASISCRSSKSLL HSNGNTYLYWFLQKPGQSPQYLIYRMSTLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPLTFGQGTKLEIK 49huAb3VH.1A/VL.1A CDR-L1 Residues 24-39 RSSKSLLHSNGNTYLY of SEQ ID NO.:56 50 huAb37VH.1A/VL.1A CDR-L2 Residues 55-61 RMSTLAS of SEQ ID NO.: 5651 huAb3VH.1A/VL.1A CDR-L3 Residues 94-102 MQHLEYPLT of SEQ ID NO.: 5652 huAb3VH.1/VL.1B VH EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMHWVRQAPGQGLEWMGYINPSSDYPNY NQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDYWGQGTTVTVSS 45 huAb3VH.1/VL.1B CDR-H1 Residues 26-35GYTFTSYTMH of SEQ ID NO.: 52 46 huAb3VH.1/VL.1B CDR-H2 Residues 50-66YINPSSDYPNYNQKFKD of SEQ ID NO.: 52 47 huAb3VH.1/VL.1A CDR-H3 Residues99-105 WGYSFDY of SEQ ID NO.: 52 57 huAb3VH.1/VL.1B VLDIVMTQSPLSLPVTPGEPASISCRSSKSLL HSNGNTYLYWYLQKPGQSPQYLIYRMSTLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPLTFGQGTKLEIK 49 huAb3VH.1/VL.1BCDR-L1 Residues 24-39 RSSKSLLHSNGNTYLY of SEQ ID NO.: 57 50huAb37VH.1/VL.1B CDR-L2 Residues 55-61 RMSTLAS of SEQ ID NO.: 57 51huAb3VH.1/VL.1B CDR-L3 Residues 94-102 MQHLEYPLT of SEQ ID NO.: 57 54huAb3VH.1B/VL.1B VH EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMHWVRQAPGQGLEWMGYINPSSDYPNY NQKFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDYWGQGTTVTVSS 45 huAb3VH.1B/VL.1B CDR-H1 Residues 26-35GYTFTSYTMH of SEQ ID NO.: 54 46 huAb3VH.1B/VL.1B CDR-H2 Residues 50-66YINPSSDYPNYNQKFKD of SEQ ID NO.: 54 47 huAb3VH.1B/VL.1B CDR-H3 Residues99-105 WGYSFDY of SEQ ID NO.: 54 57 huAb3VH.1B/VL.1B VLDIVMTQSPLSLPVTPGEPASISCRSSKSLL HSNGNTYLYWYLQKPGQSPQYLIYRMSTLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPLTFGQGTKLEIK 49huAb3VH.1B/VL.1B CDR-L1 Residues 24-39 RSSKSLLHSNGNTYLY of SEQ ID NO.:57 50 huAb37VH.1B/VL.1B CDR-L2 Residues 55-61 RMSTLAS of SEQ ID NO.: 5751 huAb3VH.1/VL.1B CDR-L3 Residues 94-102 MQHLEYPLT of SEQ ID NO.: 57 55huAb3VH.1A/VL.1B VH EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMHWVRQAPGQGLEWIGYINPSSDYPNY NQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDYWGQGTTVTVSS 45 huAb3VH.1A/VL.1B CDR-H1 Residues 26-35GYTFTSYTMH of SEQ ID NO.: 55 46 huAb3VH.1A/VL.1B CDR-H2 Residues 50-66YINPSSDYPNYNQKFKD of SEQ ID NO.: 55 47 huAb3VH.1A/VL.1B CDR-H3 Residues99-105 WGYSFDY of SEQ ID NO.: 55 56 huAb3VH.1A/VL.1B VLDIVMTQSPLSLPVTPGEPASISCRSSKSLL HSNGNTYLYWFLQKPGQSPQYLIYRMSTLASGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCMQHLEYPLTFGQGTKLEIK 49huAb3VH.1A/VL.1B CDR-L1 Residues 24-39 RSSKSLLHSNGNTYLY of SEQ ID NO.:57 50 huAb37VH.1A/VL.1B CDR-L2 Residues 55-61 RMSTLAS of SEQ ID NO.: 5751 huAb3VH.1A/VL.1B CDR-L3 Residues 94-102 MQHLEYPLT of SEQ ID NO.: 57

As described above, the CDRs of the humanized versions of the VH and VLregions of Ab3 were identical to the murine Ab3 antibody.

As Ab3 did not bind to cyno CD40 (see Example 1), humanized versions ofAb1 were selected for further analysis.

Example 3: Modification of VL CDR1 of Humanized Ab1 Antibodies

Examination of the humanized Ab1 VH and VL antibody sequences describedabove identified a potential deamidation sequence motif (an “NS” motif)exposed in the CDR1 of the light chain. The “NS” motif site that wasidentified can lead to deamidation and hydrolysis, and lead to asuccinimide-intermediate and aspartyl-ASP or iso-ASP. Thus, the sequencemotif was engineered out of the humanized Ab1 VL CDR1 sequences.Removing the “NS” motif would allow for improved antibody manufacturing.

The further engineering of humanized Ab1 resulted in six differentantibodies. Notably, four retained the antagonist activity, while twobecame agonist antibodies (huAb1v4 and huAb1v3). As shown in Table 14,huAb1v4 and huAb1v3 showed agonist activity as determined by a huCD40reporter assay, while displaying no antagonist activity as determined ina Jurkat/Reporter assay. The VH and VL amino acid sequences, as well asthe CDRs, of the variant humanized Ab1 antibodies (huAb1v1 to huAb1v6)are described below in Table 13.

TABLE 13 Humanized Ab1 antibodv variant (huAb1v#) VH and VL amino acidsequences SEQ ID Antibody NO: Clone Region Residues Amino acid sequence13 huAb1v3 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWVSYISSGRSNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 6 huAb1v3 CDR-H1 Residues 26-35 GFTFSDYGMN ofSEQ ID NO.: 13 7 huAb1v3 CDR-H2 Residues 50-66 YISSGRSNIYYADTVKG of SEQID NO.: 13 8 huAb1v3 CDR-H3 Residues 99-105 SWGYFDV of SEQ ID NO.: 13 43huAb1v3 VL DIVMTQSPDSLAVSLGERATINCKSSQSLL NLGNQKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQNDYTYPLTFGQGTKLEIK 17 huAb1v3 CDR-L1Residues 24-40 KSSQSLLNLGNQKNYLT of SEQ ID NO.: 43 11 huAb1v3 CDR-L2Residues 56-62 WASTRES of SEQ ID NO.: 43 12 huAb1v3 CDR-L3 Residues95-103 QNDYTYPLT of SEQ ID NO.: 43 15 huAb1v4 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRSNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 6 huAb1v4CDR-H1 Residues 26-35 GFTFSDYGMN of SEQ ID NO.: 15 7 huAb1v4 CDR-H2Residues 50-66 YISSGRSNIYYADTVKG of SEQ ID NO.: 15 8 huAb1v4 CDR-H3Residues 99-105 SWGYFDV of SEQ ID NO.: 15 77 huAb1v4 VLDIVMTQSPDSLAVSLGERATINCKSSQSLL NPGNQKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQNDYTYPLTFGQGTKLEIK 74 huAb1v4 CDR-L1Residues 24-40 KSSQSLLNPGNQKNYLT of SEQ ID NO.: 77 11 huAb1v4 CDR-L2Residues 56-62 WASTRES of SEQ ID NO.: 77 12 huAb1v4 CDR-L3 Residues95-103 QNDYTYPLT of SEQ ID NO.: 77 15 huAb1v5 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRSNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 6 huAb1v5CDR-H1 Residues 26-35 GFTFSDYGMN of SEQ ID NO.: 15 7 huAb1v5 CDR-H2Residues 50-66 YISSGRSNIYYADTVKG of SEQ ID NO.: 15 8 huAb1v5 CDR-H3Residues 99-105 SWGYFDV of SEQ ID NO.: 15 18 huAb1v5 VLDIVMTQSPDSLAVSLGERATINCKSSQSLL NTGNQKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQNDYTYPLTFGQGTKLEIK 19 huAb1v5 CDR-L1Residues 24-40 KSSQSLLNTGNQKNYLT of SEQ ID NO.: 18 11 huAb1v5 CDR-L2Residues 56-62 WASTRES of SEQ ID NO.: 18 12 huAb1v5 CDR-L3 Residues95-103 QNDYTYPLT of SEQ ID NO.: 18 15 huAb1v6 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRSNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 6 huAb1v6CDR-H1 Residues 26-35 GFTFSDYGMN of SEQ ID NO.: 15 7 huAb1v6 CDR-H2Residues 50-66 YISSGRSNIYYADTVKG of SEQ ID NO.: 15 8 huAb1v6 CDR-H3Residues 99-105 SWGYFDV of SEQ ID NO.: 15 43 huAb1v6 VLDIVMTQSPDSLAVSLGERATINCKSSQSLL NLGNQKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQNDYTYPLTFGQGTKLEIK 17 huAb1v6 CDR-L1Residues 24-40 KSSQSLLNLGNQKNYLT of SEQ ID NO.: 43 11 huAb1v6 CDR-L2Residues 56-62 WASTRES of SEQ ID NO.: 43 12 huAb1v6 CDR-L3 Residues95-103 QNDYTYPLT of SEQ ID NO.: 43 15 huAb1v1 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRSNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 6 huAb1v1CDR-H1 Residues 26-35 GFTFSDYGMN of SEQ ID NO.: 15 7 huAb1v1 CDR-H2Residues 50-66 YISSGRSNIYYADTVKG of SEQ ID NO.: 15 8 huAb1v1 CDR-H3Residues 99-105 SWGYFDV of SEQ ID NO.: 15 20 huAb1v1 VLDIVMTQSPDSLAVSLGERATINCKSSQSLL NRGNQKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQNDYTYPLTFGQGTKLEIK 21 huAb1v1 CDR-L1Residues 24-40 KSSQSLLNRGNQKNYLT of SEQ ID NO.: 20 11 huAb1v1 CDR-L2Residues 56-62 WASTRES of SEQ ID NO.: 20 12 huAb1v1 CDR-L3 Residues95-103 QNDYTYPLT of SEQ ID NO.: 20 13 huAb1v2 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWVSYISSGRSNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 6 huAb1v2CDR-H1 Residues 26-35 GFTFSDYGMN of SEQ ID NO.: 13 7 huAb1v2 CDR-H2Residues 50-66 YISSGRSNIYYADTVKG of SEQ ID NO.: 13 8 huAb1v2 CDR-H3Residues 99-105 SWGYFDV of SEQ ID NO.: 13 18 huAb1v2 VLDIVMTQSPDSLAVSLGERATINCKSSQSLL NTGNQKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQNDYTYPLTFGQGTKLEIK 19 huAb1v2 CDR-L1Residues 24-40 KSSQSLLNTGNQKNYLT of SEQ ID NO.: 18 11 huAb1v2 CDR-L2Residues 56-62 WASTRES of SEQ ID NO.: 18 12 huAb1v2 CDR-L3 Residues95-103 QNDYTYPLT of SEQ ID NO.: 18

In addition to the modification of the “NS” motif in the VL CDR1,variants huAb1v2 and huAb1v3 described above in Table 13 have additionalframework mutations in their VH domains.

Table 14 below provides a summary of the variant binding, agonist, andantagonist activities. Descriptions of the assays can be found above inExample 2. The “NS” motif that was mutated in the VL CDR1 is underlinedin Table 14 below. As described in Table 14, antibodies huAb1v4(containing a “P” mutation in the VL CDR1 domain) and huAb1v3(containing a “L” mutation in the VL CDR1 domain and framework mutationswithin the VH region) exhibited agonist activity despite being derivedfrom a parent antibody having antagonist activity.

TABLE 14 Sequence and Functional Summary for Variant HumanizedAntibodies VL LCDR1 Sequence Agonist: huCD40 Antagonist: Jurkat/Humanized 163- (KSSQSLLNSGNQKNYLT (SEQ Blocking of reporter assayReporter assay IC50 2.1F2.2B5 variants ID NO: 10)) SCD40L IC50 nM nMhuAb1v4 KSSQSLL NP GNQKNYLT (SEQ ID Yes 49 No NO: 74) huAb1v6 KSSQSLL NLGNQKNYLT (SEQ ID Yes No 85.0 NO: 17) huAb1v1 KSSQSLL NR GNQKNYLT (SEQ IDYes No 55 NO: 21) huAb1v2* KSSQSLL NT GNQKNYLT (SEQ ID Yes No >100 NO:19) huAb1v5 KSSQSLL NT GNQKNYLT (SEQ ID Yes No >100 NO: 19) huAb1v3*KSSQSLL NL GNQKNYLT (SEQ ID Yes 79 No NO: 17) *Additional frameworkdifferences in VH

Humanized anti-CD40 antibody huAb1v1 was selected for further study andimprovement.

Example 4: Engineering of HC CDR2 of Anti-CD40 Antibody huAb1v1

From the variants described in Example 3, antibody huAb1v1 was selectedfor further analysis. In order to further improve the potency of thisantibody, variants of the huAb1v1 heavy chain (HC) were producedcontaining mutations within the HC CDR2 domain. Seventeen additionalvariants were made (referred to as huAb1v1CDR2v1 to v17). The variant HCregions were paired with the huAb1v1 LC (SEQ ID NO: 20) for activitystudies to determine agonist and antagonist activity. Seventeen variantheavy chains were made, and in vitro activity studies showed thatgenerally the variants retained their antagonistic activity and diversepotency compared to huAb1v1. Table 15 shows that while the antibodyvariants maintained antagonistic activity, the potency of each variantvaried.

TABLE 15 Functional Summary for huAb1v1 CDR2 HC variants. EngineeredAntagonist: Jurkat/ huAb1v1CDR2 Reporter assay variants (IC50 nM)huAb1v1CDR2v17 503.8 huAb1v1CDR2v16 30.15 huAb1v1CDR2v15 339.7huAb1v1CDR2v14 21.86 huAb1v1CDR2v13 62.44 huAb1v1CDR2v12 236.1huAb1v1CDR2v11 >1000 huAb1v1CDR2v10 23.04 huAb1v1CDR2v9 7.06huAb1v1CDR2v8 >1000 huAb1v1CDR2v7 2.69 huAb1v1CDR2v6 >1000huAb1v1CDR2v5 >1000 huAb1v1CDR2v4 563.4 huAb1v1CDR2v3 218.7huAb1v1CDR2v2 >1000 huAb1v1CDR2v1 >1000

All huAb1v1 HC variants were mutated in position S55, as described belowin Table 16 (position 55 is underlined).

TABLE 16 Amino Acid Sequences of Additional VH Regions (Variants ofhuAb1v1VH) SEQ ID NO: Clone VH 15 huAb1v1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRSNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 22 huAb1v1CDR2v1EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRTNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 23huAb1v1CDR2v2 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRDNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 24 huAb1v1CDR2v3EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRENIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 25huAb1v1CDR2v4 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRRNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 26 huAb1v1CDR2v5EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRVNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 27huAb1v1CDR2v6 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRLNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 28 huAb1v1CDR2v7EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRGNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 29huAb1v1CDR2v8 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRINIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 30 huAb1v1CDR2v9EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRQNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 31huAb1v1CDR2v10 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRWNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 32 huAb1v1CDR2v11EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRMNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 33huAb1v1CDR2v1.2 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRKNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 34 huAb1v1CDR2v13EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRHNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 35huAb1v1CDR2v14 SVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRFNIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 36 huAb1v1CDR2v15EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRYNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 37huAb1v1CDR2v16 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRANIYY ADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSS 38 huAb1v1CDR2v17EVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRPNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS

Table 17 below provides a comparison of the HC CDR2 regions of theabove-described huAb1v1 variants, after engineering of the S55 residue(in bold/underlined). The VH CDR2 region of huAb1v1 corresponds to aminoacid residues 50-66 of SEQ ID NO:15.

TABLE 7 Alignment of huAb1v1 HC CDR2 variants at position 55 HC CDR2 SEQVariant VH ID 52 CDR2 NO: 50 51 52 a 53 54 55 56 57 58 59 60 61 62 63 6465 huAb1v1 7 Y I S S G R S N I Y Y A D T V K G huAb1v1CDR2v1 58 Y I S SG R T N I Y Y A D T V K G huAb1v1CDR2v2 59 Y I S S G R D N I Y Y A D T VK G huAb1v1CDR2v3 60 Y I S S G R E N I Y Y A D T V K G huAb1v1CDR2v4 61Y I S S S R R N I Y Y A D T V K G huAb1v1CDR2v5 62 Y I S S G R V N I Y YA D T V K G huAb1v1CDR2v6 63 Y I S S G R L N I Y Y A D T V K GhuAb1v1CDR2v7 42 Y I S S G R G N I Y Y A D T V K G huAb1v1CDR2v8 64 Y IS S G R I N I Y Y A D T V K G huAb1v1CDR2v9 65 Y I S S G R Q N I Y Y A DT V K G huAb1v1CDR2v10 66 Y I S S G R W N I Y Y A D T V K GhuAb1v1CDR2v11 67 Y I S S G R M N I Y Y A D T V K G huAb1v1CDR2v12 68 YI S S G R K N I Y Y A D T V K G huAb1v1CDR2v13 69 Y I S S G R H N I Y YA D T V K G huAb1v1CDR2v14 70 Y I S S G R F N I Y Y A D T V K GhuAb1v1CDR2v15 71 Y I S S G R Y N I Y Y A D T V K G huAb1v1CDR2v16 72 YI S S G R A N I Y Y A D T V K G huAb1v1CDR2v17 73 Y I S S G R P N I Y YA D T V K G

Heavy chain variable region huAb1v1CDR2v7 was selected as havingparticularly advantageous properties over the other variants that wereproduced and described above in Tables 15-17. Notably, huAb1v1CDR2v7 hasa mutation in its HC CDR2 identified as S55G. Specifically, an antibodycontaining the VL of antibody huAb1v1 (SEQ ID NO: 20; see Table 13) andVH huAb1v1CDR2v7 was determined to have a 20× increased antagonisticactivity in comparison to antibody huAb1v1.

The VL of huAbv1 and the VH of huAb1v1CDR2v7 were expressed in thecontext of two different human IgG1 constant regions. One IgG1 constantregion was selected because its effector function was diminished(hCg1.z, non-a L234A, L235A or LALA) and the other IgG1 constant regionwas selected because both its effector function was diminished and ithad a set of mutations that enhance FcRn binding (hCg1,z.non-a L234A,L235A-T250Q, M428L or LALA-QL). Tables 18 and 19 below provide the aminoacid sequence information for the heavy and light chains of anti-humanCD40 antibodies Ab101 (VL huAbv1/VH huAb1v1CDR2v7/hCg1/k-LALA) and Ab102(VL huAbv1/VH huAb1v1CDR2v7 hCg1/k-LALA-QL). Amino acid residues ofindividual CDRs of each VH or VL sequence are indicated in bold.Constant regions are underlined in Table 19.

TABLE 18 VH and VL amino acid sequences of Ab101 and Ab102 anti-hCD40antibodies. SEQ ID NO Protein region Sequence 28 Ab101 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRGNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 6 CDR-Residues 26-35 GFTFSDYGMN H1 of SEQ ID NO.: 28 42 CDR- Residues 50-66YISSGRGNIYYADTVKG H2 of SEQ ID NO.: 28 8 CDR- Residues 99-105 SWGYFDV H3of SEQ ID NO.: 28 20 Ab101 VL DIVMTQSPDSLAVSLGERATINCKSSQSLLNRGNQKNYLTWFQQKPGQPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGOGTKLEIK 21 CDR- Residues 24-40 KSSQSLLNRGNQKNYLT L1 ofSEQ ID NO.: 20 11 CDR- Residues 56-62 WASTRES L2 of SEQ ID NO.: 20 12CDR- Residues 85-93 QNDYTYPLT L3 of SEQ ID NO.: 20 28 Ab102 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMNWVRQAPGKGLEWIAYISSGRGNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARSWGYFDVWGQGTTVTVSS 6 CDR-Residues 26-35 GFTFSDYGMN H1 of SEQ ID NO.: 28 42 CDR- Residues 50-66YISSGRGNIYYADTVKG H2 of SEQ ID NO.: 28 8 CDR- Residues 99-105 SWGYFDV H3of SEQ ID NO.: 28 20 Ab102 VL DIVMTQSPDSLAVSLGERATINCKSSQSLLNRGNQKNYLTWFQQKPGQPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLTISSLQASDVAVYYCQNDYTYPLTFGQGTKLEIK 21 CDR- Residues 24-40 KSSQSLLNRGNQKNYLT L1 ofSEQ ID NO.: 20 11 CDR- Residues 56-62 WASTRES L2 of SEQ ID NO.: 20 12CDR- Residues 85-93 QNDYTYPLT L3 of SEQ ID NO.: 20

TABLE 19 Amino acid sequences of Heavy Chain (HC) and Light Chain (LC)Ab101 and Ab102 anti-hCD40 antibodies. SEQ ID Clone NO: VH Ab101-HC 39EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYG MNWVRQAPGKGLEWIAYISSGRGNIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARS WGYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK Ab101-LC 40DIVMTQSPDSLAVSLGERATINCKSSQSLLNRGN QKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYP LTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ab102-HC 41 EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNWVRQAPGKGLEWIAYISSGRGNIYYADTVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK Ab102-LC 40 DIVMTQSPDSLAVSLGERATINCKSSQSLLNRGNQKNYLTWFQQKPGQPPKLLIYWASTRESGVPDR FSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

Example 5: Functional Characterization of Humanized AntagonistAnti-hCD40 Antibodies Ab101 and Ab102 In Vitro Analysis

Humanized anti-CD40 antibodies Ab101 and Ab102 both showed antagonistactivity similar to the findings in the reporter assay described inExample 1. Since residual agonist activity is related to potentialrisks, a B cell agonist assay was developed. In this assay, theantibodies are assessed for inhibition of CD86 upregulation in human Bcells. Human B cells constitutively express CD40 and signaling throughCD40 leads to activation of B cells as measured by upregulation of CD86on the surface. B cells were activated with low dose anti-IgM and 1L4and CD40 antagonist antibodies were added. Enhancement of B cellactivation was measured as upregulation of CD86, which was observed inthe presence of agonist CD40 but not antagonist CD40 Ab suggestingundetectable agonist activity of lead candidate in vitro. To measureantagonist activity, primary human B cells were cultured withCD40L-expressing human T cell line that leads to B cell activation andupregulation of CD86 expression via CD40/CD40L interaction. Ability ofantagonist CD40 to inhibit CD86 upregulation of primary human B cellswas measured and showed strong antagonist activity of anti-CD40 antibodyAb101, as shown in FIG. 2B. FIG. 2A shows that antibody Ab101 does nothave agonist activity. Notably, as described in FIG. 2B, antibody Ab101had an IC₅₀ value of 1.337 in comparison to antagonist antibody Bib(Boehringer Ingelheim) which had an IC₅₀ value of 4.213 and agonistantibody AD11 (Astellas) which had an IC₅₀ value of 0.1906. Thus,antibody Ab101 (and Ab102 given the identical variable regions) is astrong antagonist of CD40 and shows no substantial in vitro agonistactivity.

In vivo Analysis

In order to test the in vive activity of antibody Ab101, a model ofhuman antibody generation and B cell survival was established. Briefly,when human PBMCs isolated from healthy donors were transferred intoimmunocompromised scid mice, the generation of human IgG in response tomouse antigens was measurable 14 days later. Additionally. FACS analysisof splenocytes from these mice indicated human B cell engraftment andsurvival. An antigen specific response was measured by including achallenge with tetanus toxoid (TetTox) vaccine and measuring anti-TetToxspecific IgG (Naito, 2000; Jeurissen, 2004).

Treatment of these huscid mice with weekly doses of anti-human CD40(Ab101, 5 mg/kg IP) resulted in >85% inhibition of human IgG production(FIG. 3A) and B cell survival (FIG. 3B), clearly demonstrating theantibody is active in vivo. Specifically. FIG. 3A shows that antibodyAb101 was able to inhibit IgG production in comparison to the Igcontrol, and FIG. 3B indicates that administration of antibody Ab101 inthe above huscid model inhibited B cell survival.

Example 6: Epitope Analysis of Fab Ab102

Using Fab Ab102, crystallography studies were performed to determine theepitope to which Fab Ab101 binds. As described above, the VH and VLsequences of antibodies Ab101 and Ab102 are the same, and, therefore,the use of Fab Ab102 in the following crystal structure study isrepresentative of the binding features of both antibodies Ab101 andAb102.

Crystal structures were determined for Ab102 Fab alone and for Ab101 Fabcomplexed to CD40 antigen. Crystals were obtained and data was collectedat the IMCA-CAT 17ID beamline. A crystal structure of Ab102 Fab wassolved to 1.74 Å resolution and the Ab102 Fab/CD40 complex structure wassolved to 2.84 Δ resolution. The crystal structures provided theidentification of the 3D conformational epitope of Ab102 Fab.

Identification of 3D Conformational Epitope of Ab102 Fab

The contacts between Ab102 Fab and CD40 involve both critical hydrogenbond and hydrophobic interactions which stabilize the interface. A listof molecular contacts (measuring under 4.0 Å) were generated using theprogram NCONT in the CCP4 suite of programs. The contacts were measuredbetween the two separate crystallographic CD40 monomers and thecorresponding bound light and heavy chains of the Ab102 Fab. Additionalcontacts were observed between the Ab102 Fab and a crystallographic CD40dimer (the dimer created by crystal contacts). Based on this informationthe epitope of Ab102 Fab binding comprises of the topographical regiondefined by Cys62-Phe67, Gln79-Cys83, Arg90-Thr99. Thr24-Cys37 of CD40.

Materials and Methods Preparation and Purification of CD40 Antigen:

A DNA sequence encoding the human CD40 extracellular domain (amino acids1-193) was cloned into pHybE vector followed by an in-frame C-terminalTev protease cleavage site and hexahistidine tag (SEQ ID NO: 115). Theplasmid was transfected into HEK293 6e cells (MRL) at 1×10e6 cells/mlusing the transfection reagent Polyethylenimine (PEI, Polysciences Inc)at a PEI:DNA ratio of 4:1. The transfected cell culture was fed withtryptone-N1 (to 0.5%) at 24 h post-transfection. On day 7post-transfection, the transfected cell culture was cleared bycentrifugation followed by filtration through 0.2u PES filter (Corning).Cleared medium was buffer exchanged to PBS, pH 7.4 using a Kvick TFFsystem equipped with 10 kDa membranes (GE Healthcare) and loaded on a 5ml HisTrap FF column (GE Healthcare) equilibrated with PBS, pH 7.4. Thecolumn was washed with 25 mm imidazole in PBS, pH 7.4 and bound proteinwas eluted with 250 mM imidazole in PBS, pH 7.4. Eluted protein wasconcentrated using Amicon Ultra-5 centrifugal filter devices (Millipore)with 10 kDa molecular weight cut-off, and further purified by SEC on a26/60 Superdex 200 column (GE Healthcare) equilibrated and run with PBS.pH 7.4. Fractions containing CD40 were pooled, concentration measured byabsorbance at 280 nm, and samples analyzed by SEC. SDS-PAGE, and massspectrometry. [CD40(h)(21-193)]-Tev-His6 (“His6” disclosed as SEQ ID NO:115) was stored in aliquots at −80° C.

Preparation and Purification of CD40 Ab102 Fab Fragment:

Fab fragment of CD40 Ab102 was prepared by papain cleavage of the parentmAb as detailed below. Papain was activated with 50 mM cysteine in PBS,pH 7.4 buffer. mAb CD40 Ab102 [hu IgG1/k] LALA QL in PBS. pH 7.4 bufferwas mixed with papain at 1:100 weight ratio of papain to mAb andincubated for 1 h at 37° C. The reaction was quenched with 5 mMiodoacetamide. The mixture was purified on 10 ml Mab SelectSure resin(GE Healthcare) where the Fab fragment was collected as flow through.The flow through was concentrated using an Ultrafree-15 Biomax 10 kDamolecular weight cut-off (MWCO) centrifugal device (Millipore). Theconcentrated mixture was purified on 2.6 cm×60 cm Sephacryl 200 HiPrepcolumn (GE Healthcare) pre-equilibrated in 50 mM HEPES, 50 mM NaCl. pH7.5 buffer. Fractions containing Fab fragment (monitored by UVabsorbance at 280 nm) were pooled and frozen at −80° C. Sample puritywas assessed by analytical SEC. SDS-PAGE and mass spectrometry.

CD40/CD40 Ab102 Fab Complex Preparation:

Recombinant human CD40 was expressed in mammalian expression system andsubsequently purified using techniques well known in the art.Recombinant human CD40 and CD40 Ab102 Fab protein were mixed at a 1.1:1molar ratio and incubated for 4 h at 4° C. The complex sample was loadedonto a 2.6 cm×60 cm Sephacryl 200 HiPrep column (GE Healthcare)pre-equilibrated in 50 mM HEPES. 50 mM NaCl. pH 7.5 buffer at 1 ml/min.

Fractions containing the complex (monitored by UV absorbance at 280 nm)were pooled and concentrated to 18 mg/ml using an Ultrafree-15 Biomax 10kDa molecular weight cut-off (MWCO) centrifugal device (Millipore).Sample purity was assessed by analytical SEC and SDS-PAGE.

Ab102 Fab Crystallization:

Fab alone was supplied at 22.5 mg/ml in 50 mM HEPES, 50 mM NaCl, pH 7.5.Crystals grew by vapor diffusion at 23° C. The reservoir contained 25%(w/v) PMME 550, 0.1M MES pH 6.5, 0.01M zinc sulfate. The drop was madeby adding equal volumes of protein and reservoir solution. Crystals grewas thick prisms and were cryo-protected using the reservoir solutionwith the addition of 10% (v/v) propylene glycol. Crystals wereharvested, swished through cryo-solution and cryo-cooled directly inliquid nitrogen. Diffraction data to 1.74 Å were collected under gaseousnitrogen at 100 K at the 171D beamline at the Advanced Photon Source atArgonne National Laboratories (Argonne IL).

Ab102 Fab Complexed to CD40 Antigen Crystallization:

The Fab complex was supplied at 18 mg/ml in 50 mM HEPES, 50 mM NaCl, pH7.5. The antigen construct used was [CD40 (h) (21-193)]-TEV-6His (“His6”disclosed as SEQ ID NO: 115). Crystals grew by vapor diffusion at 23° C.The reservoir contained 2M ammonium sulfate, 0.1 M phosphate-citrate pH4.2. The drop was made by adding equal volumes of protein and reservoirsolution. Crystals grew as thin rods and were cryo-protected using 2.5Mlithium sulfate. Crystals were harvested, swished through cryo-solutionand cryo-cooled directly in liquid nitrogen. Diffraction data to 2.84 Åwere collected under gaseous nitrogen at 100 K at the 171D beamline atthe Advanced Photon Source at Argonne National Laboratories (ArgonneIL).

Structure Determination of Ab102 Fab and Ab102 Fab CD40 Complex

Diffraction data for both crystal structures were processed using theprogram autoPROC from Global Phasing Ltd.

The Ab102 Fab dataset was processed in the space group C222₁ with thefollowing unit cell dimensions; a=64.65 b=130.4 c=132.6. A maximumlikelihood molecular replacement solution was determined using theprogram PHASER using an Fab search model reported previously (ProteinData Bank entry 3Q0S). Coordinates for 1 Fab molecule were generatedbased on the molecular replacement solution. Preliminary refimement ofthe resulting solution was conducted using REFMAC and the programBUSTER. Iterative protein model building was conducted using the programCOOT and examination of 2Fo-Fc and Fo-Fc electron-density maps.Refinement concluded with the addition of water molecules using BUSTER.Final refinement statistics reported R_(free)/R_(work) values of0.23/0.19.

The Ab102 Fab CD40 complex dataset was processed in the space groupP2₁2₁2 with the following unit cell dimensions: a=173.3 b=76.0 c=126.1.A maximum likelihood molecular replacement solution was determined usingthe program PHASER using the previously solved Ab102 Fab reported above.Coordinates for 2 Fab molecules were found based on the molecularreplacement solution. Preliminary refinement of the resulting solutionwas conducted using REFMAC and the program BUSTER. The model for CD40was built manually using the program COOT and examination of 2Fo-Fc andFo-Fc electron-density maps. Refinement concluded with the addition ofwater molecules using BUSTER. Final refinement statistics reportedR_(free)/R_(work) values of 0.25/0.20.

Example 7: Neutralization Potency and Agonist Activity of Ab102 in theMonocyte Activation Assay

The following methods were used in this example which examined theantagonist and agonist activity of Ab102 in vitro.

Antagonist Assay:

The ability of Ab102 to block CD40-mediated monocyte activation wasassessed in an antagonist assay. Purified monocytes were mixed with 1μg/mL MEGACD40L (Enzo) in the presence of 80 ng/mL GM-CSF and 80 ng/mLIFNγ at a concentration of 2×10⁶/mL. 50 μL was added per well in a96-well U-bottom tissue culture (TC) plate. Dilutions of testedmaterials were prepared in culture medium, and 50 μL of dilutions wereadded to human monocytes obtained from a donor. Cells were cultured at37° C. 5% CO₂ for two days before supernatants were harvested forcytokine (TNF) analysis using Meso Scale Discovery (MSD) immunoassayplatform.

Agonist Assay:

The ability of Ab102 to induce monocyte activation through CD40 wasassessed. MEGACD40L (Enzo) was used as a positive control for monocyteactivation. Purified human monocytes were diluted to 2×10⁶ mL in culturemedium in the presence of 80 ng/mL GM-CSF and 80 ng/mL IFNγ, and 50μL/well was added in a 96-well U bottom TC plate. Dilutions of testedmaterials were prepared in culture medium, and 50 μL of dilutions wereadded to the monocytes. Cells were cultured at 37° C. 5% CO₂ for twodays before supernatants were harvested for cytokine (TNF) analysisusing Meso Scale Discovery (MSD) immunoassay platform.

As myeloid cells play an important role in the pathogenesis of Crohn'sdisease, the above-mentioned monocyte-based assays were developed toevaluate the functional activity of Ab102. CD40 signaling induces theactivation of monocytes and thereby the production of inflammatorycytokines such as TNF. Representative monocyte antagonist and agonistassays for Ab102 are shown in FIGS. 5A and 5B, respectively. As shown inFIG. 5A, Ab102 blocked the expression of TNF in aconcentration-dependent manner. In the agonist assay format, solubleCD40L induced the production of TNF from monocytes with an EC₅₀ of 1.9nM, while Ab102 did not induce TNF production at concentrations up to200 nM. As described in FIG. 5B, Ab102 levels of TNF were similar tothose of the negative control (non-relevant IgG) showing little to nodetectable TNF production. Consistent results were obtained from threedifferent donors with the IC50 values shown in Table 20 below.

TABLE 20 Summary of Functional Assessment of Ab102 in MonocyteActivation Assay Reagent Experiment Antagonist IC₅₀ (nM) Ab102 1 0.06 20.23 3 0.11 Average ± SD 0.13 ± 0.08

In sum, results from testing Ab102 in both the antagonist and theagonist assays described above showed that Ab102 is an antagonistanti-CD40 antibody that is substantially free of agonist activity withno measurable agonist activity.

Example 8: Cross-Reactivity of Ab102

The cross-reactivity of Ab102 with CD40 from various species was tested.Using standard techniques, Alexa 647 labeled Ab102 demonstrated similarbinding kinetics to both human and cynomolgus monkey CD40 on the surfaceof B cells with an EC50 value of 0.89±0.17 nM on human cells and1.4±0.15 nM on cynomolgus monkey cells, as shown in Table 21, below.Binding of Ab102 to mouse, rat, and rabbit CD40 could not be detected atconcentrations up to 30 μg/mL (200 nM).

TABLE 21 Summary of Ab102 Binding to Various CD40 on the Surface of BCells Reagent Species EC₅₀ (nM) Ab102 Human 0.89 ± 0.17 Monkey  1.4 +0.15 Mouse ND Rat ND Rabbit ND

In sum, Ab102 bound to human and cynomolgus monkey CD40, but showed nodetectable binding to mouse, rat, or rabbit CD40 using standard bindingassays.

Example 9: Use of Mouse Anti-CD40 Antibody 138 to Treat T-Cell TransferColitis

The following methods were used in an in vivo study to determine theability of a mouse anti-CD40 antibody (antibody 138) to treat colitis.Anti-murine CD40 antibody 138 has similar characteristics to Ab102,e.g., antibody 138 is an antagonist antibody with no substantial agonistactivity like Ab102. Thus, antibody 138 is representative of Ab102activity in the mouse model of Example 9. The following describes an invivo T cell transfer model of colitis.

Isolation and Injection of Naïve T Cells

On Day 0, spleens were collected from balb/c mice and placed in RPMImedia supplemented with 4% fetal bovine serum (complete media) on ice. Asingle cell suspension was obtained by mechanical disruption and passingthrough a 100 μm cell strainer into RPMI media supplemented with 4%fetal bovine serum (complete media). Cells were collected bycentrifugation (1250 rpm for 10 minutes at 4° C.) and re-suspension inRobosep Buffer (Stem Cell Technologies). Cell concentration was measuredusing a Moxi Mini Cell Counter (Orflo) and adjusted to 1×10⁸ cells/mL inRobosep buffer. CD4+ T cells were isolated using a negative selectionmagnetic bead kit (Stem Cell Technologies) according to themanufacturer's directions. Cells were further purified by FACS to yieldpopulations of CD4⁺CD45RB^(high) and CD4⁺CD45RB^(low) which werecollected as the brightest 42% and the dullest 12% of cells,respectively. Cells were counted and adjusted to 1×10⁶ cells/ml, and 0.5mL (1×10⁵ cells) were injected intraperitoneally (IP) into SCID mice.

Treatment

Mouse anti-CD40 antibody 138 was then administered at various doses IPin PBS twice/week to SCID mice (described above) starting either at thetime of cell injection (prophylactic treatment) or after disease wasconfirmed by endoscopy (therapeutic treatment). In addition, a number ofcontrol groups were included. Groups received either 1) 15 mg/kg IP ofantibody 951 (a non-relevant IgG) in PBS twice/week, or 2) antibody 138(an antibody that blocks CD40L) IP in PBS, twice/week. Additionally, inthe T-cell transfer (TCT) studies, either an anti-p40(IL-12/23) antibodyor an anti-TNF monoclonal antibody was administered as clinicallyrelevant control comparators.

Endoscopy

At various times following cell injection into the mice, disease wasassessed by colonoscopy. Following anesthesia with isoflurane, aflexible gavage needle was slowly inserted into the anus and 300 μL PBSwas slowly injected to remove fecal pellets. Animals were allowed torecover from anesthesia and ambulate to facilitate passing of anyremaining pellets (approximately five minutes). Mice were againanesthetized with isoflurane and the endoscopy probe (Karl Storz) wasinserted into the anus to a depth of 3 cm. Photo images were captured at3, 2 and 1 cm from the anal verge. Images were scored at a later timeusing the scale detailed in Table 22 below. Scores for each parameter ateach of the three distances were combined to produce the MurineEndoscopic Disease Activity Index (MEDAI) Sum Score, shown in Table 22.The maximum score that could be obtained using the MEDAI Sum Score was24.

In the case of therapeutic dosing, endoscopic scoring three weeks aftercell injection was used to confirm disease and group animals fortreatment.

TABLE 22 MEDAI Endoscopic Scoring Parameter Score Description Exudate 0Normal 1 covers <50% of colon circumference 2 covers >50% of coloncircumference Vascularity 0 Normal 1 vessels disconnected, small vesselsnot visible 2 large vessels not visible, starburst pattern 3 surfacebleeding apparent, vessels appear leaky Mucosal 0 Normal Granularity 1slight cobblestone appearance 2 pronounced and extensive cobblestoneappearance 3 mucosal protrusion, reduced lumen

Histology

GI samples were submitted in cassettes in the stretch segmented wholecolon orientation, which allowed for analysis of the entire colon lengthand processed for formalin-fixed paraffin-embedded (FFPE). Blocks weresectioned at 5 micron, and mounted on glass slides prior to performingimmunohistochemistry to detect ionized calcium binding adaptor molecule1 (IBA1) using anti-IBA1 antibody (Cat. No. 019-19741; Wako PureChemical Industries, Ltd.). The IBA1 marker was used to identifymacrophages in the tissue sections. Slides were counterstained withmethyl green, dehydrated, and mounted with a glass coverslip.

Slides were then scanned at 4× by the Vectra imaging system and 4× lowpower mosaic images were reviewed prior to selection of areas to imageat 20×.

Vectra imagining rescanned slides and captured the selected 20× highpower high resolution images which were then subjected to image analysisalgorithms in the inForm software (Perkin Elmer). The inForm algorithmset that was used has three algorithms: the first thresholds thestaining for IBA1 and CD3 in spectrally imaged files, to eliminatebackground/extraneous stain. The subsequent algorithms segment thetissue into tissues of interest (lamina propria, epithelium, muscularis,submucosa and background), and quantifies either CD3 or IBA1 in the RGBimages produced from the first algorithm.

InForm data was exported to text files which were merged into singledata files for either tissue segmentation or cell segmentation data.

Based on the validation of antagonist activity in the acute models, amodel of chronic colitis was used to establish proof of concept. Threestudies were conducted to test the effect of blocking colitis followingtransfer of naïve T cells into an immunocompromised host (SCID mouse). Asingle dose level was investigated in both prophylactic and therapeutictreatment mode while a full dose response was examined in prophylacticmode.

Dose Response of a Mouse Anti-CD40 Antibody 138 Administered at the Timeof Cell Transfer

The dose response of prophylactically administered mouse anti-CD40antibody 138 was determined using the T cell transfer model of colitis.Treatment that covered a range of doses (0.5, 1.5, 5 and 15 mg/kg) wasinitiated at the time of cell transfer. Doses down to 1.5 mg/kg resultedin maximum inhibition of the MEDAI sum score, while 0.5 mg/kg had nosignificant effect. Activity of the mouse anti-CD40 antibody 138 wasequivalent to the standard dose of anti-p40IL-12/23 treatment used as apositive control. Histological analysis of colonic sections showed adecrease in macrophages (a general measure of inflammation) thatcorrelated well with endoscopic assessment of disease, as described inFIG. 6 and FIG. 7. Specifically. FIG. 6 describes dose responseinhibition of endoscopy score with prophylactic administration of mouseanti-CD40 antibody 138 at day 39 (terminal score). In FIG. 6, RBlowrefers to the negative control group. CD45RBlow cells do not mediatedisease. In this model of colitis, disease is mediated by CD45RBhi cellsthat were transferred to animals. FIG. 7 shows the number of IBA1+macrophages in the colon of the mice (determined histologically) anddescribes lower levels of macrophages at doses greater than 0.5 mg/kgand lower than the p40 positive control Levels of circulating mouseanti-CD40 antibody 138 were measured 96 hours after the final dose(equivalent to C_(trough)) and were shown to be dose responsive asdescribed in FIG. 8. The lowest dose level (0.5 mg/kg) resulted in aconcentration <lower limit of quantification (LLOQ) in all but oneanimal in that group.

Dose Response of a Mouse Anti-CD40 Antibody 138-Administered 3 WeeksPost Cell Transfer

When mouse anti-CD40 antibody 138 treatment was initiated three weekspost cell injection, following confirmation of endoscopic disease, adose responsive inhibition of the MEDAI sum score was observed with thehighest dose (15 mg/kg) reaching statistical significance (FIG. 9A).Histological analysis of IBA1⁺ macrophages in the colon as a measure ofmyeloid inflammation yielded similar results, as described in FIG. 9B.The mean serum concentration of antibody 138 measured 72 hours after thefinal dose was 191.9 μg/ml in animals that received 15 mg/kg.

Example 10: T-Cell Dependent Antibody Responses in Cynomolgus Monkeys

A T-cell dependent antibody response (TDAR) assay was conducted incynomolgus monkeys. Two/sex group were administered Ab102 at dosages of0 (vehicle only) or 10 mg/kg subcutaneously (SC) for 5 weeks. Keyholelimpet hemocyanin (KLH) was administered to all animals on Day 8. Serumsamples were collected from each animal at −11, −7, 0, 4, 7, 10, 14 and21 days relative to KLH administration. All animals were returned totesting facility animal colony following completion of last scheduledblood collection (FIG. 10).

It was found that Ab102 suppressed both the anti-KLH IgM and IgGantibody T cell dependent antibody responses, as compared to vehicleonly treated animals, as shown in FIG. 10.

The findings in this study were consistent with the pharmacologicsuppression of CD40-dependent IgM and IgG antibody production followingparenteral administration of a prototypic foreign protein. The resultssuggested that use of Ab102 may be relevant for treating lupus, whereautoantibody production is part of the disease. These findings alsosupported the biologic relevance of cynomolgus monkeys as an appropriatespecies for preclinical toxicology studies. Further, the studydemonstrated cross-reactivity to cynomolgus monkey CD40, and shows invivo activity of Ab102 in a mechanism (T-dependent antibody response)which is known to require CD40.

Example 11: Use of Mouse Anti-CD40 Antibody 138 to Treat Systemic LupusErythematosus (SLE)

Because of the antagonist activity of the mouse anti-CD40 antibody 138that was shown in the above acute models and in a model of colitis (seeExample 9), the efficacy of this mouse anti-CD40 antibody was examinedin mouse models of systemic lupus erythematosus (SLE). Two SLE modelswere used: MRL/lpr and NZB/W-F1 (described in Theofilopoulos and Kono.1999. Murine lupus models: gene-specific and genome-wide studies. InLahita R. G., ed., Systemic Lupus Erythematosus, 3rd edn, p. 145). Therationale for assessing the efficacy of anti-CD40 treatment in MRL/lprand NZB/W-F1 mice is two-fold. First, the models differ in theirmanifestations of SLE. NZB/W-F1 mice spontaneously develop lupusnephritis and sialadenitis, whereas MRL/lpr mice develop joint and skinmanifestations in addition to nephritis and sialadenitis (Andrews et al.J. Exp. Med. 148: 1198-1215, 1978). Since patients differ in theirmanifestations of SLE, the use of both models allows the assessment ofpotential efficacy in a majority of SLE patients. Second, MRL/lpr andNZB/W-F1 mice differ in the genetic basis for their disease (Perry etal. J. Biomed. Biotech. 2011. Article ID 271694), and therefore,efficacy across models increases confidence in the translation togenetically heterogeneous human.

11.1. MRL/Lpr Model of SLE

To determine efficacy, mice were administered mouse anti-CD40 antibody138 by intraperitoneal injection (i.p.) beginning at 10 weeks of age atthe doses indicated in Table 23, below. PBS injections were used as anegative control, and prednisolone was used as a positive control in thestudy.

TABLE 23 Group n Treatment Dose 1 18 PBS 2x/wk i.p. 2 18 Antibody 138 15mg/kg 2x/wk i.p. 3 18 Antibody 138 5 mg/kg 2x/wk i.p. 4 18 Antibody 1381.5 mg/kg 2x/wk i.p. 5 18 Antibody 138 15 mg/kg 1x/wk i.p. 6 18Prednisolone 10 mg/kg PO sid

Proteinuria was monitored weekly by Albustix (a brand of urine dipsticks used to test for urine protein). High proteinuria, defined as≥300 mg/dL, began to develop shortly after treatment began, as shown inFIG. 11A. As described in FIG. 11A, by study completion at day 63,50-60% of untreated PBS control mice and mice treated with 1.5 mg/kg ofanti-CD40 antibody had developed high proteinuria. In contrast, nearlyall mice in the other treatment groups maintained low proteinuriathroughout the study. The 15 mg/kg 1× per week treatment group wassignificantly different from the PBS control. Survival was alsomonitored and it was found that dosing at 15 mg/kg 1× per week and 5mg/kg 2× per week significantly extended survival over the untreated PBScontrol animals, as shown in FIG. 11B. It should be noted that many micewere euthanized due to distress caused by lymphadenopathy caused by theFaslpr mutation before they developed nephritis. Thus, the observeddecrease in survival cannot be attributed solely to nephritis.Nevertheless, these data indicate that anti-CD40 antibody dosedependently prevented proteinuria and extended survival of lupus-proneMRL/lpr mice.

Efficacy of the anti-CD40 antibody for treating SLE was also evaluatedin hematoxylin and eosin (H&E) stain stained formalin-fixedparaffin-embedded (FFPE) tissue sections from the kidney, salivarygland, and ankle joints of mice from the various treatment groups.Severity of disease in all test organs in the control untreated PBS miceincreased over the 9 weeks of the study as the MRL mice aged from 10weeks of age at the beginning to 19 weeks at the end.

In the kidney, anti-CD40 antibody treatment was efficacious whenadministered at 15 mg/kg in reducing glomerular disease at both 29 and63 days of treatment, as shown in FIG. 12A. As glomerular diseaseseverity worsened in aging MRL mice, anti-CD40 antibody treatmentmaintained efficacy at minimizing glomerular disease at 5 and 15 mg/kg.Anti-CD40 antibody given in a dose of 15 mg/kg once a week was aseffective as dosing twice a week. Anti-CD40 antibody given at a dose of5 mg/kg twice a week was also near the same effectiveness. Anti-CD40antibody given at a dose of 5 and 15 mg/kg was effective at reducingperivascular (PV) infiltrates in the kidney at 29 and 63 days, as shownin FIG. 12B, with a trend at reducing tubulointerstitial (TI) early indisease, as shown in FIG. 12C.

In the salivary gland, anti-CD40 antibody given at a dose of 1.5, 5 and15 mg/kg was efficacious in reducing salivary gland inflammation at day29, while 15 mg/kg maintained efficacy at day 63 of therapy (FIG. 13A).Salivary gland infiltration did not change significantly in theuntreated mice after day 29.

In the tarsal joint tissue, anti-CD40 antibody administered doses of1.5, 5 and 15 mg/kg was efficacious in reducing inflammation around thejoint at day 29, while 15 mg/kg maintained efficacy at day 63 of therapy(FIG. 13B). In the joints, inflammation trended lower in the 19 weekmice compared to the 10 week mice. Nevertheless, anti-CD40 antibodytreatment significantly reduced inflammation to near zero at 15 mg/kg at63 days of therapy.

Germinal center (GC) formation requires B and T cell interaction throughengagement of CD40 on the GC B cell with CD40L on follicular helper Tcells (Tfh). GCs are the anatomical structures where plasma cells andmemory B cells are generated, and are where affinity maturation and Igclass switch occur. They are critical to the development of highaffinity and pathogenic autoantibodies in SLE.

The mouse anti-CD40 antibody 138 disrupted the B and T cell interactionand prevented GC formation. To assess the extent to which GC formationwas prevented, the number of GC B cells and Tfh cells in the spleen wasdetermined by flow cytometry (FIG. 14). Tfh cell numbers weresignificantly lower than controls at day 29 in all anti-CD40 antibody138 treated mice regardless of dose. Among the doses tested, the 5 mg/kgdose group remained significantly lower than control at day 63 (FIG. 14,see panels (i) and (ii)). GC B cells were also lower than control at day29 at all anti-CD40 antibody doses, and they remained lower at day 63 inmice receiving anti-CD40 antibody at doses of 5 and 15 mg/kg. However,these differences from untreated controls were not statisticallysignificant. Notwithstanding the results, there was an overall trend inthe data toward a therapeutic effect in all dose groups. Furthermore, inthese experiments, the murine anti-CD40 antibody demonstrated little tono agonist activity.

Circulating total IgG levels, in addition to the levels of autoreactiveantibodies, increase over time in murine and human SLE. Therefore, toassess the effect of anti-CD40 antibody 138 on antibody production,total circulating IgM and IgG levels were examined. In addition,anti-dsDNA antibodies, a common lupus-associated autoantibody, were alsoexamined. It was found that the total IgG levels in mice treated withanti-CD40 antibody at doses of 15 and 1.5 mg/kg were significantly lowerthan those in untreated controls at day 29, as described in FIG. 15A.Significantly lower total IgG levels persisted up to day 63 in micetreated with anti-CD40 antibody 138 at 15 mg/kg, as described in FIG.15B. In addition, significantly lower levels were observed in micetreated with anti-CD40 antibody 138 at 5 mg/kg at this time point. Nodifference was found in circulating IgM levels.

Anti-dsDNA titers were not significantly different in anti-CD40 antibody138 treated and untreated control mice at day 29, as described in FIG.16A. However, by day 63 anti-dsDNA titers had increased substantially inuntreated control mice, but declined in mice treated with 15 and 5 mg/kganti-CD40 antibody 138, although the difference was not significant, asdescribed in FIG. 16B.

The results obtained from the above study (Example 11.1) indicate thatanti-CD40 antibody 138 is efficacious in preventing the development ofnephritis in the lupus-prone MRL/lpr mice. In addition, anti-CD40antibody 138 prevented the development of salivary gland and jointinflammation. This study suggests that the antagonist mouse anti-CD40antibody 138, having similar properties to Ab102, is efficacious fortreating human SLE.

11.2. NZB/W-F₁ Mouse Model of SLE

A second mouse model for SLE was also tested to determine if antagonistmouse anti-CD40 antibody 138 is effective for treatment of the disease.Specifically, to determine the efficacy of anti-CD40 antibody 138, theantibody was tested in NZB/W-F1 mice, where both prophylactic andtherapeutic regimens were used according to the dosing scheduledescribed in Table 24, below. As in study 11.1, PBS served as a negativecontrol and prednisolone was a positive control.

TABLE 24 Group n Treatment Bose Prophylactic 1 20 PBS i.p 2x/wk 2 20Antibody 138 15 mg/kg i.p. 2x/wk 3 20 Antibody 138 1.5 mg/kg i.p. 2x/wk4 20 Antibody 138 15 mg/kg i.p. 1x/wk 5 20 Prednisolone 10 mg/kg PO sidTherapeutic 6 13 PBS i.p. 2x/wk 7 12 Antibody 138 15 mg/kg i.p. 2x/wk 812 Prednisolone 10 mg/kg PO sid

For the prophylactic regimen, mice began treatment at 26 weeks of age.All mice were verified to have <300 mg/dL protein. For the therapeuticregimen, a rolling enrollment was used; untreated mice were monitoredweekly for proteinuria and were enrolled into one of the 3 arms of thetherapeutic regimens when they developed proteinuria of ≥300 mg/dL.

Prophylactic Treatment

Proteinuria was monitored weekly and as shown in FIG. 17A, where about50% of untreated control mice were proteinuric by 32 weeks of age. Incontrast, only 1 of the anti-CD40 antibody 138 treated mice and none ofthe prednisolone treated mice developed high proteinuria. These resultswere significant from untreated controls. Survival, as shown in FIG.17B, mirrored the proteinuria finding with all treatments significantlydifferent from PBS controls. Thus, both the 15 and 1.5 mg/kg treatmentdoses prevented proteinuria and extended survival.

Therapeutic Treatment

Therapeutic treatment with anti-CD40 antibody 138 was also efficaciousin this second mouse SLE model. As shown in FIG. 18A and FIG. 18B, micetreated with anti-CD40 antibody 138 developed low proteinuria over time,whereas neither the untreated control mice nor the prednisolone treatedmice developed low proteinuria. Based on the rate of recovery fromproteinuria, as shown in FIG. 18B, it is estimated that the average timeto recovery of proteinuria was 23±7 days. Anti-CD40 antibody 138treatment also significantly extended survival, as described in FIG.18C.

Saliva Output

Saliva production was measured in both prophylactically andtherapeutically treated mice to assess salivary gland function.Anesthetized mice were administered pilocarpine nitrate and over an 8minute period saliva was collected on a cotton swab. Saliva output wasmeasured as the net weight increase of the cotton swab. In theprophylactic study, it was found that saliva production by untreatedcontrol mice was highly variable, but significantly different from thatof NZBWF-1 non-diseased younger mice, as described in FIGS. 19A and 19B.The variability was likely due to the level of disease since themajority of vehicle control mice with the lowest saliva production wereproteinuric (FIGS. 19A and 19B, purple vs black in vehicle group).Importantly, however, saliva production by anti-CD40 antibody 138treated mice was comparatively uniform (FIG. 19A) and significantlygreater than in untreated control mice. Although all anti-CD40 antibody138 treated cohorts had higher saliva production, only the 1.5 mg/kgtreated cohort retained significance when measured as a function of bodyweight (FIG. 19B). Nevertheless, these data indicate that prophylacticanti-CD40 antibody 138 treatment can prevent the loss of salivary glandfunction.

In therapeutically treated mice, it was found that anti-CD40 antibody138 treated mice had significantly higher saliva production thanuntreated controls (FIGS. 20 A and 20B). FIGS. 20 A and 20B show thatsaliva production was preserved by therapeutic dosing of anti-CD40antibody. This was evident whether considering total saliva or salivanormalized for body weight. Notably, the untreated mice were allproteinuric, whereas none of the anti-CD40 antibody treated mice wereproteinuric. It can therefore be concluded that therapeutic dosing of anantagonist anti-CD40 antibody that is substantially free of agonistactivity, prevents or reverses the decline in salivary gland function.

This study indicates that antagonist anti-CD40 antibody 138 wasefficacious in preventing the development of nephritis in thelupus-prone NZB/W-F1 mice and in rescuing these mice from nephritis. Inaddition, anti-CD40 antibody prevented the development of salivary glandand joint inflammation. This study supports the hypothesis that anantagonist anti-CD40 antibody that is substantially free of agonistactivity will be efficacious in human SLE.

Methods used in Examples 11.1 and 11.2 included the following:

MRL/Lpr Mice

MRL/lpr: Anti-CD40 antibody (antibody 138) was administered i.p. to 10week old MRL/pr mice in one of 4 doses: 15 mg/kg. 5 mg/kg, or 1.5 mg/kgtwice per week, or 15 mg/kg once per week. Mice treated with PBS(vehicle) i.p. twice per week were included as a negative control, andmice treated with 10 mg/kg prednisolone PO sid were included as apositive control.

NZB/W-F₁: Anti-CD40 antibody 138 was administered to NZB/W-F, mice inboth prophylactic and therapeutic regimens. For the prophylacticregimen, mice were given anti-CD40 i.p. beginning at 26 weeks of age ineither of two doses, twice per week at 15 mg/kg or 1.5 mg/kg.

Mice given prednisolone at 10 mg/kg PO sid or given PBS served aspositive and negative controls, respectively. Mice were tested forproteinuria at the outset and any mice with ≥300 mg/dL were excludedfrom the prophylactic study. For the therapeutic regimen, mice begantreatment as they developed proteinuria of 2300 mg/dL. Mice receivedanti-CD40 i.p. at 15 mg/kg twice per week. Mice given prednisolone at 10mg/kg PO sid, or PBS served as positive and negative controls,respectively.

Proteinuria

Urine was tested weekly for protein level using Albustix reagent strips(Siemens 2191, Pittsburgh, Pa.). Mice were considered proteinuric whenurine protein levels increased to ≥300 mg/dL for at least 2 consecutivetests or prior to death or euthanasia.

Flow Cytometry

Splenic single cell suspensions were made in Hanks buffered saltsolution (Invitrogen) with 1% heat inactivated fetal bovine serum(Invitrogen) and 1× penicillin/streptomycin (Sigma, St Louis, Mo.).Erythrocytes were removed by centrifugation and the cells resuspended instaining buffer (PBS (Invitrogen) with 1.5% heat inactivated fetalbovine serum and 0.02% sodium azide (Sigma)). Cells were stained withantibodies against CD3 (145-2011, BD), CD4 (GK1.5 eBioSciences), ICOS(C398.4A, Biolegend), CXCR5 (L138D7, Biolegend), PD-1 (29F, 1A12,Biolegend). GL7 (A488, Biolegend), CD19 (BUV395, BD), CD95 (Jo2,Biolegend). Antibodies were directly couple to fluoresceinisothicyanate, phycoerythrin, phycoerythrin and cyanine 5,allophycocyanin, peridinin chlorophylla and cyanine 5.5, or biotin.Total B cells were identified as CD19+; GC B cells as CD19+, CD95+, andGL7⁺: Tfh cells as CD3+, CD4+, ICOS+, CXCR5+, and PD-1+. Cells wereanalyzed by a FACSCalibur (BD Biosciences) flow cytometer and analyzedwith FlowJo software (version 8.5, Treestar Inc.).

Histology

As vehicle control mice became moribund, kidney, ankle and salivarygland tissues were collected and fixed in 10% neutral buffered formalin.In addition, at specific intermediate time points these same tissues andblood were collected from representative mice of each group. Tissueswere fixed in 10% neutral-buffered formalin for 8 hours, processed andparaffin-embedded for H&E. Inflammatory infiltrates were evaluated inthe kidney, salivary gland, and ankle based on evaluation of routine H&Estained formalin-fixed paraffin embedded (FFPE) sections. For thekidney, a pathologist scored 3 um H&E sections on a 0 to 4 scale forglomerular disease, perivascular infiltration, and tubulointerstitialinfiltration based on the following scoring criteria: Glomerulardisease: 0=no disease; I=segmental thickening of mesangium in occasionalglomeruli: 2=segmental to diffuse thickening of mesangium in mostglomeruli; 3=diffuse thickening of the mesangium, hypercellularity,enlarged podocytes, no adhesions; 4=diffuse thickening of the mesangiumwith areas worse than others with one or more of the following:coagulated proteins, fibrosis, hypocellularity, enlarged podocytes,adhesions and crescents of Bowman's capsule. Perivascular renalinflammation: 0=up to a few rare lymphocytes; 1=few lymphocytes formingloose aggregates; 2=lymphocytes forming discrete small aggregates;3=polarized aggregate of lymphocytes that bulge into the lumen of theadjacent vein but fail to fully surround the arcuate artery;4=lymphocyte aggregates fully surrounding the arcuate artery withoutpolarization. Tubulointerstitial (TI) infiltration: 0=no infiltrates;1=minimal to mild TI infiltrates; 2=mild infiltrates between 20% oftubules; 3=mild to moderate infiltrates between up to 50% of tubules;4=moderate infiltrates between and surrounding >50% tubules throughoutrenal cortex. Salivary and joint inflammation scores were based on thesame principles with a 0-4 score of increasing infiltrates within thetissue.

Saliva Output

To measure salivary gland output, mice were first sedated withoxygen/isoflurane in a small isolation chamber. After sedation mice werei.p. administered 30 ul (60 ug) of pilocarpine nitrate (Sigma). Twominutes after injection, pre-weighed children's safety swabs (trimmed to˜1.5 cm, cotton plus stem) were placed in the animals mouth behind thefront teeth. Mice were placed on their sides to allow pooling of salivain the cheek and adsorption by the cotton swab. After 8 minutes theswabs were removed and weighed to calculate the net saliva weight.

ELISA

Total circulating IgG and IgM levels were determined by ELISA usingeBioscience kits (cat #88-50400, 88-50470) according to themanufacturer's instructions.

SEQUENCE SUMMARY Sequence Identifier Protein Sequence SEQ ID NO: 1 HumanCD40 MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDT WNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSD TICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILL VLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ SEQ ID NO: 2 Human Ig gamma-1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE constant regionPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQOGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 3 Human Ig gamma-1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE constant regionPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT mutantVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK SEQ ID NO: 4 HumanIg Kappa TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE constant regionAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 5 Variable heavy EVQLVESGGGLVKPGGSLKVSCAASGFTFSDYGMN chainof: WVRQAPEKGLEWIAYISSGRSNIYYADTVKGRFTI Ab1 (murine)SRDNAKNTLFLQMTSLRSEDTAMYYCARSWGYFDV (CDRs in bold) WGTGTTVTVSS SEQ IDNO: 6 Heavy chain CDR1 GFTFSDYGMN of: Ab1 (murine) Ab2 (murine)huAb1v1CDR2v1 to huAb1v1CDR2v17 huAb1v1 huAb1v5 huAb1v6 huAb1v4 huAb1v3Ab101 Ab102 SEQ ID NO: 7 Heavy chain CDR2 YISSGRSNIYYADTVKG of:Ab1(murine) huAb1VH.1/VL.1 huAb1v1 huAb1v5 huAb1v3 huAb1v4 SEQ ID NO: 8Heavy chain CDR3 SWGYFDV of: Ab2 (murine) Ab1(murine) huAb1VH.1/VL.1huAb1VH.1A/VL.1 huAb1VH.1A/VL.1A huAb1v1 huAb1v1CDR2v1 to huAb1v1CDR2v17huAb1v5 huAb1v6 huAb1v2 huAb1v3 huAb1v4 Ab101 Ab102 SEQ ID NO: 9Variable light DIVMTQSPSSLTVTAGEMVTMSCKSSQSLLNSGNQ chain of:KNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFAG Ab1(murine)SGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFG AGTKLEIK SEQ ID NO: 10 Light chainCDR1 KSSQSLLNSGNQKNYLT of: Ab2 (murine) Ab1 (murine) huAb1VH1/VL.1huAb1VH1A/VL.1 huAb1VH1A/VL.1A SEQ ID NO: 11 Light chain CDR2 WASTRESof: Ab1 (murine) Ab2 (murine) huAb1VH.1A/VL.1A huAb1VH.1/VL.1AhuAb1VH.1/VL.1 huAb1VH.1A/VL.1 huAb1v5 huAb1v2 huAb1v6 huAb1v1 huAb1v3huAb1v4 Ab101 Ab102 SEQ ID NO: 12 Light chain CDR3 QNDYTYPLT of: Ab1(murine) Ab2 (murine) huAb1VH.1A/VL.1A huAb1VH.1/VL.1A huAb1VH.1/VL.1huAb1VH.1A./VL.1 huAb1v5 huAb1v2 huAb1v6 huAb1v1 huAb1v3 huAb1v4 Ab101Ab102 SEQ ID NO: 13 Variable heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMNchain of: WVRQAPGKGLEWVSYISSGRSNIYYADTVKGRFTI huAb1VH.1/VL.1SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV huAb1VH.1/VL.1A WGQGTTVTVSS huAb1v2huAb1v3 SEQ ID NO: 14 Variable light DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQchain of: KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSG huAb1VH.1/VL.1SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFG huAb1VH.1A./VL.1 QGTKLEIK SEQ ID NO:15 Variable heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain of:WVRQAPGKGLEWIAYISSGRSNIYYADTVKGRFTI huAb1VH.1A/VL.1SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV huAb1VH.1A/VL.1A WGQGTTVTVSS huAb1v1huAb1v5 huAb1v4 huAb1v6 SEQ ID NO: 16 Variable lightDIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQ chain of:KNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSG huAb1VH.1A/VL.1ASGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFG huAb1VH.1/VL.1A QGTKLEIK SEQ ID NO:17 Light chain CDR1 KSSQSLLNLGNQKNYLT of: huAb1v6 huAb1v3 SEQ ID NO: 18VL DIVMTQSPDSLAVSLGERATINCKSSQSLLNTGNQ huAb1v5KNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSG huAb1v2SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFG (CDRs in bold) QGTKLEIK SEQ ID NO:19 Light chain CDR1 KSSQSLLNTGNQKNYLT of: huAb1v5 huAb1v2 SEQ ID NO: 20Variable light DIVMTQSPDSLAVSLGERATINCKSSQSLLNRGNQ chain (VL)of:KNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSG huAb1v1SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFG Ab101 QGTKLEIK Ab102 (CDRs in bold)SEQ ID NO: 21 Variable light KSSQSLLNRGNQKNYLT chain CDR1 of: huAb1v1Ab101 Ab102 SEQ ID NO: 22 Variable HeavyEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRTNIYYADTVKGRFTI huAb1v1CDR2v1SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 23 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRDNIYYADTVKGRFTI huAb1v1CDR2v2SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 24 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRENIYYADTVKGRFTI huAb1v1CDR2v3SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 25 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRRNIYYADTVKGRFTI huAb1v1CDR2v4SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 26 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRVNIYYADTVKGRFTI huAb1v1CDR2v5SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 27 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRLNIYYADTVKGRFTI huAb1v1CDR2v6SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 28 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRGNIYYADTVKGRFTI huAb1v1CDR2v7SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV Ab101 WGQGTTVTVSS Ab102 (CDRs inbold) SEQ ID NO: 29 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN huAb1v1CDR2v8WVRQAPGKGLEWIAYISSGRINIYYADTVKGRFTI (CDRs in bold)SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV WGQGTTVTVSS SEQ ID NO: 30 VariableHeavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRQNIYYADTVKGRFTI huAb1v1CDR2v9SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 31 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRWNIYYADTVKGRFTI huAb1v1CDR2v10SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 32 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRMNIYYADTVKGRFTI huAb1v1CDR2v11SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 33 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRKNIYYADTVKGRFTI huAb1v1CDR2v12SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 34 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRHNIYYADTVKGRFTI huAb1v1CDR2v13SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 35 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRFNIYYADTVKGRFTI huAb1v1CDR2v14SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 36 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRYNIYYADTVKGRFTI huAb1v1CDR2v15SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 37 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRANIYYADTVKGRFTI huAb1v1CDR2v16SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 38 Variable Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN chain(VH) of:WVRQAPGKGLEWIAYISSGRPNIYYADTVKGRFTI huAb1v1CDR2v17SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (CDRs in bold) WGQGTTVTVSS SEQ IDNO: 39 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN sequenceWVRQAPGKGLEWIAYISSGRGNIYYADTVKGRFTI Ab101SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV (constant regionWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA is underlined;LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS CDRs in bold)SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ IDNO: 40 Light chain DIVMTQSPDSLAVSLGERATINCKSSQSLLNRGNQ sequenceKNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSG Ab101SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFG Light chainQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVC sequenceLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK Ab102DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP Humanized VTKSFNRGEC (constantregion is underlined; CDRs in bold) SEQ ID NO: 41 Heavy chainEVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMN sequenceWVRQAPGKGLEWIAYISSGRGNIYYADTVKGRFTI Ab102SRDNAKNSLYLQMNSLRAEDTAVYYCARSWGYFDV HumanizedWGQGTTVTVSSASTKGPSVFPLAPSKSTSGGTAA (constant regionLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS is underlined;SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV CDRs in bold)DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK PKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK SEQ ID NO: 42 Heavy chain CDR2YISSGRGNIYYADTVKG of: Ab2 huAb1v1CDR2v7 Ab101 Ab102 SEQ ID NO: 43Variable light DIVMTQSPDSLAVSLGERATINCKSSQSLLNLGNQ chain of:KNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSG huAb1v6SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFG huAb1v3 QGTKLEIK (CDRs in bold) SEQID NO: 44 Variable heavy QVQLQQSGAELARPGASVKMSCKAFGYTFTSYTMH chain of:WVKQRPGQGLEWIGYINPSSDYPNYNQKFKDKATL Ab3 (murine)TADKSSSTAYMQLSSLTSEDSAVYYCARWGYSFDY (CDRs in bold) WGQGTTLTVSS SEQ IDNO: 45 Heavy chain GYTFTSYTMH CDR1 of: Ab3 (murine) huAb3VH.1/VL.1huAb3VH.1/VL.1A huAb3VH.1/VL.1B huAb3VH.1B/VL.1 huAb3VH.1B/VL.1AhuAb3VH.1B/VL.1B huAb3VH.1A/VL.1 huAb3VH.1A/VL.1A huAb3VH.1A/VL.1B SEQID NO: 46 Heavy chain YINPSSDYPNYNQKFKD CDR2 of: Ab3 (murine)huAb3VH.1/VL.1 huAb3VH.1/VL.1A huAb3VH.1/VL.1B huAb3VH.1B/VL.1huAb3VH.1B/VL.1A huAb3VH.1B/VL.1B huAb3VH.1A/VL.1 huAb3VH.1A/VL.1AhuAb3VH.1A/VL.1B SEQ ID NO: 47 Heavy chain WGYSFDY CDR3 of: Ab3 (murine)huAb3VH.1/VL.1 huAb3VH.1/VL.1A huAb3VH.1/VL.1B huAb3VH.1B/VL.1huAb3VH.1B/VL.1A huAb3VH.1B/VL.1B huAb3VH.1A/VL.1 huAb3VH.1A/VL.1AhuAb3VH.1A/VL.1B SEQ ID NO: 48 Variable lightDIVMTQAAPSVSVIPGESVSISCRSSKSLLHSNGN chain of:TYLYWFLQRPGQSPQYLIYRMSTLASGVPDRFSGS Ab3 (murine)GSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGA (CDRs in bold) GTKLELK SEQ ID NO: 49Light chain RSSKSLLHSNGNTYLY CDR1 of: Ab3 (murine) huAb3VH.1/VL.1huAb3VH.1B/VL.1 huAb3VH.1A/VL.1 huAb3VH.1/VL.1A huAb3VH.1B/VL.1AhuAb3VH.1A/VL.1A huAb3VH.1/VL.1B huAb3VH.1B/VL.1B huAb3VH.1A/VL.1B SEQID NO: 50 Light chain RMSTLAS CDR2 of: Ab3 (murine) huAb3VH.1/VL.1huAb3VH.1B/VL.1 huAb3VH.1A/VL.1 huAb3VH.1/VL.1A huAb3VH.1B/VL.1AhuAb3VH.1A/VL.1A huAb3VH.1/VL.1B huAb3VH.1B/VL.1B huAb3VH.1A/VL.1B SEQID NO: 51 Light chain MQHLETPLT CDR3 of: Ab3 (murine) huAb3VH.1/VL.1huAb3VH.1B/VL.1 huAb3VH.1A/VL.1 huAb3VH.1/VL.1A huAb3VH.1B/VL.1AhuAb3VH.1A/VL.1A huAb3VH.1/VL.1B huAb3VH.1B/VL.1B huAb3VH.1A/VL.1B SEQID NO: 52 Variable heavy EVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMH chain of:WVRQAPGQGLEWMGYINPSSDYPNYNQKFKDRVTI huAb3VH.1/VL.1TADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDY huAb3VH.1/VL.1A WGQGTTVTVSShuAb3VH.1/VL.1B SEQ ID NO: 53 Variable lightDIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGN chain of:TYLYWYLQKPGQSPQLLIYRMSTLASGVPDRFSGS huAb3VH.1/VL.1GSGTDFTLKISRVEAEDVGVYYCMQHLEYPLTFGQ huAb3VH.1B/VL.1 GTKLEIKhuAb3VH.1A/VL.1 (CDRs in bold) SEQ ID NO: 54 Variable heavyEVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMH chain of:WVRQAPGQGLEWMGYINPSSDYPNYNQKFKDRVTL huAb3VH.1B/VL.1TADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDY huAb3VH.1B/VL.1A WGQGTTVTVSShuAb3VH.1B/VL.1B (CDRs in bold) SEQ ID NO: 55 Variable heavyEVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYTMH chain of:WVRQAPGQGLEWIGYINPSSDYPNYNQKFKDRATL huAb3VH.1A/VL.1TADKSTSTAYMELSSLRSEDTAVYYCARWGYSFDY huAb3VH.1A/VL.1A WGQGTTVTVSShuAb3VH.1A/VL.1B (CDRs in bold) SEQ ID NO: 56 Variable lightDIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGN chain of:TYLYWFLQKPGQSPQYLIYRMSTLASGVPDRFSGS huAb3VH.1/VL.1AGSGTDFTLKISRVEAEDVGVYYCMQHLEYPLTFGQ huAb3VH.1B/VL.1A GTKLEIKhuAb3VH.1A/VL.1A (CDRs in bold) SEQ ID NO: 57 Variable lightDIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGN chain of:TYLYWYLQKPGQSPQYLIYRMSTLASGVPDRFSGS huAb3VH.1/VL.1BGSGTDFTLKISRVEAEDVGVYYCMQHLEYPLTFGQ huAb3VH.1B/VL.1B GTKLEIKhuAb3VH.1A/VL.1B (CDRs in bold) SEQ ID NO: 58 Heavy chain CDR2YISSGRTNIYYADTVKG of: huAb1v1CDR2v1 SEQ ID NO: 59 Heavy chain CDR2YISSGRDNIYYADTVKG of: huAb1v1CDR2v2 SEQ ID NO: 60 Heavy chain CDR2YISSGRENIYYADTVKG of: huAb1v1CDR2v3 SEQ ID NO: 61 Heavy chain CDR2YISSGRRNIYYADTVKG of: huAb1v1CDR2v4 SEQ ID NO: 62 Heavy chain CDR2YISSGRVNIYYADTVKG of: huAb1v1CDR2v5 SEQ ID NO: 63 Heavy chain CDR2YISSGRLNIYYADTVKG of: huAb1v1CDR2v6 SEQ ID NO: 64 Heavy chain CDR2YISSGRINIYYADTVKG of: huAb1v1CDR2v8 SEQ ID NO: 65 Heavy chain CDR2YISSGRQNIYYADTVKG of: huAb1v1CDR2v9 SEQ ID NO: 66 Heavy chain CDR2YISSGRWNIYYADTVKG of: huAb1v1CDR2v10 SEQ ID NO: 67 Heavy chain CDR2YISSGRMNIYYADTVKG of: huAb1v1CDR2v11 SEQ ID NO: 68 Heavy chain CDR2YISSGRKNIYYADTVKG of: huAb1v1CDR2v12 SEQ ID NO: 69 Heavy chain CDR2YISSGRHNIYYADTVKG of: huAb1v1CDR2v13 SEQ ID NO: 70 Heavy chain CDR2YISSGRFNIYYADTVKG of: huAb1v1CDR2v14 SEQ ID NO: 71 Heavy chain CDR2YISSGRYNIYYADTVKG of: huAb1v1CDR2v15 SEQ ID NO: 72 Heavy chain CDR2YISSGRANIYYADTVKG of: huAb1v1CDR2v16 SEQ ID NO: 73 Heavy chain CDR2YISSGRFNIYYADTVKG of: huAb1v1CDR2v17 SEQ ID NO: 74 Light chain CDR1KSSQSLLNPGNQKNYLT of: huAb1v4 SEQ ID NO: 75 Variable heavyEVQLVESGGGLVKPGGSLKVSCAASGFTFSDYGMN chain of:WVRQSPEKGLEWIAYISSGRGNIYYADTVKGPFTI Ab2 (murine)SRDNAKNTLFLQMTSLRSEDTAMYYCARSWGYFDV (CDRs in bold) WGTGTTVTVSS SEQ IDNO: 76 Variable light DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQ chain of:KNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFTG Ab2(murine)SGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFG (CDRs in bold) AGTKLELK SEQ ID NO:77 Variable light DIVMTQSPDSLAVSLGERATINCKSSQSLLNPGNQ chain of:KNYLTWFQQKPGQPPKLLIYWASTRESGVPDRFSG huAb1v4SGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFG QGTKLEIK SEQ ID NO: 78 ConsensusG(F/Y)TF(S/T)(D/S)Y(G/T)M(N/H) sequence of variable heavy chain CDR1 SEQID NO: 79 Consensus YI(S/N)(S/P)(G/S)(R/S)(D/S/G)(N/Y) sequence of(I/P)(Y/N)Y(A/N)(D/Q)(T/K)(V/F)K variable heavy (G/D) chain CDR2 SEQ IDNO: 80 Consensus (S/W)(W/G)(G/Y)(Y/S)FDV sequence of variable heavychain CDR3 SEQ ID NO: 81 Human Ig LambdaQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG constant regionAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSY LSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSEQ ID NO: 82 Human heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFT acceptorsequence VH1-18&JH6 FR1 21/28&JH4 FR1 SEQ ID NO: 83 Human heavy chainWVRQAPGQGLEWMG acceptor sequence VH1-18&JH6 FR2 SEQ ID NO: 84 Humanheavy RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR chain acceptor sequenceVH1-18&JH6 FR3 SEQ ID NO: 85 Human heavy WGQGTTVTVSS chain acceptorsequence VH1-18&JH6 FR4 VH2-26&JH6 FR4 VH1-46&JH6 FR4 SEQ ID NO: 86Human heavy WVRQAPGQRLEWMG chain acceptor sequence 21/28&JH4 FR2 SEQ IDNO: 87 Human heavy RVTITRDTSASTAYMELSSLRSEDTAVYYCAR chain acceptorsequence 21/28&JH4 FR3 SEQ ID NO: 88 Human heavy WGQGTLVTVSS chainacceptor sequence 21/28&JH4 FR4 M60&JH4 FR4 SEQ ID NO: 89 Human heavyQVTLKESGPVLVKPTETLTLTCTVSGFSLS chain acceptor sequence VH2-26&JH6 FR1SEQ ID NO: 90 Human heavy WIRQPPGKALEWLAH chain acceptor sequenceVH2-26&JH6 FR2 SEQ ID NO: 91 Human heavyRLTISKDTSKSQVVLTMTNMDPVDTATYYCAR chain acceptor sequence VH2-26&JH6 FR3SEQ ID NO: 92 Human heavy QVTLRESGPALVKPTQTLTLTCTLYGFSLS chain acceptorsequence M60&JH4 FR1 SEQ ID NO: 93 Human heavy WIRQPPGKALEWLA chainacceptor sequence M60&JH4 FR2 SEQ ID NO: 94 Human heavyRLTISKDTSKNQVVLTMTNMDPVDTATYYCAR chain acceptor sequence M60&JH4 FR3 SEQID NO: 95 Human heavy RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR chain acceptorsequence VH1-46&JH6 FR3 SEQ ID NO: 96 Human lightDIOMTQSPSSLSASVGDRVTITC chain acceptor sequence A20&JK4 FR1 SEQ ID NO:97 Human light WYQQKPGKVPKLLIY chain acceptor sequence A20&JK4 FR2 SEQID NO: 98 Human light GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC chain acceptorsequence A20&JK4 FR3 SEQ ID NO: 99 Human light FGGGTKVEIKP chainacceptor sequence A20&JK4 FR4 III-3R&JK4 FR4 A1&JK4 FR4 SEQ ID NO: 100Human light GVPSRISGSGSGTDFTFTISSLQPEDIATYYC chain acceptor sequenceIII-3R&JK4 FR3 SEQ ID NO: 101 Human light DVVMTQSPLSLPVTLGQPASISC chainacceptor sequence A1&JK4 FR1 SEQ ID NO: 102 Human light WFQQRPGQSPRRLIYchain acceptor sequence A1&JK4 FR2 SEQ ID NO: 103 Human lightGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC chain acceptor sequence A1&JK4 FR301&JK2 FR3 SEQ ID NO: 104 Human light DIVMTQTPLSLPVTPGEPASISC chainacceptor sequence 01&JK2 FR1 SEQ ID NO: 105 Human light WYLQKPGQSPQLLIYchain acceptor sequence 01&JK2 FR2 SEQ ID NO: 106 Human lightFGQGTKLEIKR chain acceptor sequence 01&JK2 FR4 SEQ ID NO: 107 Human CD40EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFT extracellularETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLR domainVQQKGTSETDTICTCEEGWHCTSEACESCV SEQ ID NO: 108 Consensus(K/R)SS(Q/K)SLL(N/H)S(G/—)N(Q/G) sequence of (K/N)(N/T)YL(T/Y) variablelight chain CDR1 SEQ ID NO: 109 Consensus (W/R)(A/M)ST(R/L)(E/A)Ssequence of variable light chain CDR2 SEQ ID NO: 110 Consensus(Q/M)(N/Q)(D/H)(Y/L)(T/E)YPLT sequence of variable light chain CDR3 SEQID NO: 111 Heavy chain YISSGRXNIYYADTVKG CDR2 Where “X” is any aminoacid other than T, D, V, L, I, K SEQ ID NO: 112 Heavy chainYISSGRXNIYYADTVKG CDR2 Where “X” is any amino acid SEQ ID NO: 113 Lightchain KSSQSLLNXGNQKNYLT CDR1 where “X” is not amino acid residue Pro SEQID NO: 114 Human light WYQQKPGKAPKLLIY chain acceptor sequence III-3R&JK4 FR2 SEQ ID NO: 115 Histidine tag His His His His His His

1. An isolated nucleic acid encoding an antagonist anti-CD40 antibodyamino acid sequence comprising a) a heavy chain comprising an amino acidsequence as set forth in SEQ ID NO: 41, and a light chain comprising anamino acid sequence as set forth in SEQ ID NO: 40; b) a heavy chain CDR1comprising an amino acid sequence as set forth in SEQ ID NO:6, a heavychain CDR2 comprising an amino acid sequence as set forth in SEQ IDNO:42, a heavy chain CDR3 comprising an amino acid sequence as set forthin SEQ ID NO:8, a light chain CDR1 comprising an amino acid sequence asset forth in SEQ ID NO:21, a light chain CDR2 comprising an amino acidsequence as set forth in SEQ ID NO:11, and a light chain CDR3 comprisingan amino acid sequence as set forth in SEQ ID NO:12; or c) a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 28 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 20
 2. A vector comprising the isolatednucleic acid of claim
 1. 3. A host cell comprising the vector of claim2.
 4. A method of producing an antagonist anti-CD40 antibody, or antigenbinding portion thereof, the method comprising the steps of culturingthe host cell of claim 3 in culture medium under conditions sufficientto produce the antagonist anti-CD40 antibody, or antigen binding portionthereof.
 5. A method reducing human CD40 activity, the method comprisingthe step of contacting human CD40 with an anti-CD40 antibody, orantigen-binding portion thereof, such that human CD40 activity isreduced, wherein the anti-CD40 antibody is an isolated antibody, orantigen binding portion thereof, that binds an epitope of human CD40defined by the topographic regions Cys62-Phe67, Gln79-Cys83,Arg90-Thr99, and Thr24-Cys37 of SEQ ID NO:1.
 6. The method of claim 5,wherein the antibody, or antigen binding portion thereof, is humanized.7. The method of claim 5, wherein the antibody, or antigen bindingportion thereof, is substantially free of agonist activity.
 8. Themethod of claim 5, wherein the anti-CD40 antibody is an antagonistanti-CD40 antibody, or antigen-binding portion thereof, comprising aheavy chain CDR1 comprising an amino acid sequence as set forth in SEQID NO:6, a heavy chain CDR2 comprising an amino acid sequence as setforth in SEQ ID NO:42, a heavy chain CDR3 comprising an amino acidsequence as set forth in SEQ ID NO:8, a light chain CDR1 comprising anamino acid sequence as set forth in SEQ ID NO:21, a light chain CDR2comprising an amino acid sequence as set forth in SEQ ID NO:11, and alight chain CDR3 comprising an amino acid sequence as set forth in SEQID NO:
 12. 9. The method of claim 5, wherein the anti-CD40 antibody isan antagonist anti-CD40 antibody, or antigen-binding portion thereof,comprising a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 28 and a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO:
 20. 10. Themethod of claim 5, wherein the anti-CD40 antibody is an anti-CD40antibody comprising a heavy chain comprising an amino acid sequence asset forth in SEQ ID NO: 41, and a light chain comprising an amino acidsequence as set forth in SEQ ID NO:
 40. 11. The method of claim 5,wherein the method comprises reducing human CD40 activity in a humansubject having a disorder in which CD40 activity is detrimental, themethod comprising the step of administering to the human subject ananti-CD40 antibody, or antigen binding portion thereof, such that humanCD40 activity in the human subject is reduced, wherein the anti-CD40antibody, or antigen-binding portion thereof, is an isolated antibody,or antigen binding portion thereof, that binds an epitope of human CD40defined by the topographic regions Cys62-Phe67, Gln79-Cys83,Arg90-Thr99, and Thr24-Cys37 of SEQ ID NO:1. 12.-13. (canceled)
 14. Themethod of claim 11, wherein the anti-CD40 antibody is an antagonistanti-CD40 antibody, or antigen-binding portion thereof, comprising aheavy chain CDR comprising an amino acid sequence as set forth in SEQ IDNO:6, a heavy chain CDR2 comprising an amino acid sequence as set forthin SEQ ID NO:42, a heavy chain CDR3 comprising an amino acid sequence asset forth in SEQ ID NO:8, a light chain CDR1 comprising an amino acidsequence as set forth in SEQ ID NO:21, a light chain CDR2 comprising anamino acid sequence as set forth in SEQ ID NO:11, and a light chain CDR3comprising an amino acid sequence as set forth in SEQ ID NO:
 12. 15. Themethod of claim 11, wherein the anti-CD40 antibody is an antagonistanti-CD40 antibody, or antigen-binding portion thereof, comprising aheavy chain variable domain comprising an amino acid sequence set forthin SEQ ID NO: 28 and a light chain variable domain comprising an aminoacid sequence set forth in SEQ ID NO:
 20. 16. The method of claim 11,wherein the anti-CD40 antibody is an anti-CD40 antibody comprising aheavy chain comprising an amino acid sequence as set forth in SEQ ID NO:41, and a light chain comprising an amino acid sequence as set forth inSEQ ID NO:
 40. 17.-22. (canceled)
 23. A method of determining thepresence of CD40 or fragment thereof in a test sample by an immunoassay,wherein the immunoassay comprises contacting the test sample with atleast one anti-CD40 antibody, or antigen-binding portion thereof, and atleast one detectable label, wherein the anti-CD40 antibody, orantigen-binding portion thereof, is an isolated antibody, or antigenbinding portion thereof, that binds an epitope of human CD40 defined bythe topographic regions Cys62-Phe67, Gln79-Cys83, Arg90-Thr99, andThr24-Cys37 of SEQ ID NO:1.
 24. The method of claim 22, wherein theantibody, or antigen binding portion thereof, is humanized.
 25. Themethod of claim 22, wherein the antibody, or antigen binding portionthereof, is substantially free of agonist activity.
 26. The method ofclaim 22, wherein the anti-CD40 antibody is an antagonist anti-CD40antibody, or antigen-binding portion thereof, comprising a heavy chainCDR1 comprising an amino acid sequence as set forth in SEQ ID NO:6, aheavy chain CDR2 comprising an amino acid sequence as set forth in SEQID NO:42, a heavy chain CDR3 comprising an amino acid sequence as setforth in SEQ ID NO:8, a light chain CDR1 comprising an amino acidsequence as set forth in SEQ ID NO:21, a light chain CDR2 comprising anamino acid sequence as set forth in SEQ ID NO:11, and a light chain CDR3comprising an amino acid sequence as set forth in SEQ ID NO:
 12. 27. Themethod of claim 22, wherein the anti-CD40 antibody is an antagonistanti-CD40 antibody, or antigen-binding portion thereof, comprising aheavy chain variable domain comprising an amino acid sequence set forthin SEQ ID NO: 28 and a light chain variable domain comprising an aminoacid sequence set forth in SEQ ID NO:
 20. 28. The method of claim 22,wherein the anti-CD40 antibody is an anti-CD40 antibody comprising aheavy chain comprising an amino acid sequence as set forth in SEQ ID NO:41, and a light chain comprising an amino acid sequence as set forth inSEQ ID NO: 40.